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A Dream of Red Mansions tells the story that a stone at the foot of Blue Ridge Peak returns from dynamic state to static state. Through this story, Tsao Hsueh-chin showed us how to pacify the mind so as to resolve the trouble and pain of life , and then gained freedom of spirit and tranquility of soul. Pacifying the mind experiences two processes: the entrance of principle and the entrance of practice. The former refers to theoretically elucidate, while the latter means practically doing. The entrance of practice, dominated by induction and dispelling, further divided into four steps; Starting off in the Void (which is truth) came to the contemplation of Form (which is illusion) ; and from Form engendered Passion; and by communicating Passion entered again into Form;and from Form awoke to the Void. These four steps made the 120-chapter novel become an organic whole. Taking the seventy-fifth chapter as partition, the first-half of the novel describes blossoming of predestined fate,along with induction and dispelling of stone's desire for enjoying the honor and wealth in the mortal world. The rest part depicts fruiting of predestined fate, together with induction and dispelling of stone's sentiment for decline and withering. [ABSTRACT FROM AUTHOR]

Objective: To analyze effect of accordion technique on bone mineralization of extended bone segment in treating tibial bone defect with bone transport., Methods: From May 2017 to October 2019, 22 patients with tibial bone defects were treated with Ilizarov bone-transport technique, and divided into two groups after bone-transport was completed, 11 patients in each group. In observation group, there were 9 males and 2 females aged from 20 to 60 years old with an average of (42.6± 13.3) years old;the length of bone defect ranged from 3 to 13 cm with an average of(6.4±2.6) cm;2 patients were suffered from upper tibial bone defects, 3 patients were middle and 6 patients were lower;patients were treated with accordion technique for 35 days. In control group, there were 10 males and 1 female aged from 41 to 60 years old with an average of (51.6±6.4) years old;the length of bone defect ranged from 3 to 10.7 cm with an average of (6.6±2.5) cm;1 patient was suffered from upper tibial bone defects, 3 patients were middle and 7 patients were lower;patients were treated with lock external fixator to waiting bone mineralization. The content of hydroxyapatite (HAP) extended bone segment was measured after bone-transport completed immediately, 35, 65 and 95 days after bone-transport was completed, respectively, then the mineralization time and healing time were compared between two groups, and the therapeutic effect of bone defect was evaluated by using Paley scoring criteria., Results: Twenty-two patients were followed up from 18 to 36 months with an average of (27.0±6.3) months. The wounds on the bone defects healed spontaneously during bone transport, and there were no wound complications such as skin infection or skin necrosis occurred. There were statisticaldifference in the content of HAP of the extended bone segments at 35, 65 and 95 days after bone-transport between two groups ( P <0.05). There were difference in mineralization time and healing time between two groups ( P <0.05). According to Paley standard evaluation, 10 patients got excellent results, and 1 good in observation group; while 9 patients got excellent results, 2 good in control group;and there was no differences between two groups ( Z =-0.607, P = 0.544)., Conclusion: Accordion technique and locking external fixator mineralization in prolonging bone segment healing after bone-transport have the equal clinical effect, while the accordion technique could significantly accelerate the growth rate of HAP and shorten the mineralization time and healing time.

Objective: To explore the clinical effect of acupoint puncture combined with Ilizarov technique in the treatment of knee osteoarthritis in the elderly., Methods: From March 2015 to February 2016, 76 patients with primary knee osteoarthritis were treated with tibial osteotomy acupoint puncture grouop and Ilizarov technique anatomical puncture group, including 24 males and 52 females, aged 56 to 75 years old with an average of 61.4 years old, and a course of 3 to 17 years with an average of 5.2 years. Among them, 38 cases were treated with external fixation of acupoint puncture needle and 38 cases were treated with external fixation of anatomical puncture needle. Preoperative full-length X-ray of both lower limbs showed tibial varus deformity, narrowing of medial knee joint space and enlargement of lateral knee joint space. The force line of the affected knee and lower limb was moved inward by body surface measurement, and the KSS knee function score was decreased. Symptoms included medial knee pain, flexion and extension, and conservative treatment for more than 2 years., Results: The lower limb force lines of both groups were corrected and the osteotomy ends healed well. No nonunion of osteotomy, inadequate correction of lower limbs or recurrence of deformity were found. Seventy-five patients were followed up for 3, 6, 12 and 24 months after operation. There was no significant difference in knee joint mobility between the two groups before operation and on 6, 12, 24 months after operation( F =1.346, P >0.05). There were significant difference in KSS pain and total score between the two groups at 3 months after operation, acupoint puncture group was better than anatomical puncture group( P <0.05); there was no significant difference in KSS score at 12 months after operation( P >0.05)., Conclusions: The acupoint puncture group formed a potential acupuncture effect in the acupoint area by continuously tightening the steel needle on Ilizarov ring external fixator during the post-operative adjustment. Within three months after wearing external fixator, the knee pain symptoms of knee osteoarthritis were relieved rapidly, continuously and effectively, which was significantly better than that of the anatomical puncture group., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© 2019 by the China Journal of Orthopaedics and Traumatology Press.)

Objective: To study the taxonomy and distribution of Chinese medicinal centipedes., Methods: The species of Chinese medicinal centipedes were investigated in the light of their morphology. According to the feature of life, the distribution of centipedes were explored., Results: There were 12 centipede species in China, and seven of them were used for medical, the species could be effectively distinguished by the identification key. It was suggested that their characteristics were related to the climatic factors such as temperature, humidity, altitude and air pressure., Conclusion: The distribution of medicinal centipede is characteristics of "three river system distribution belts" and "three geographical distribution areas". The results provide the basis for the development and application of medicinal centipedes in China.

Objective: To explore the surgical method and clinical curative effect of Ilizarov technique combined with limited surgical treatment for neurotrophic malnutrition equinovarus accompanied with weight-bearing area ulcer., Methods: From July 2004 to December 2011, 21 cases of neurological disorders equinovarus with skin ulcer in weight-bearing area were treated including 14 males and 7 females with an average age of 21.3 years old ranging from 8 to 32 years. Among them,19 cases with talipes equinovarus were on account of spina bifid and 2 cases on account of spinal cord injury of lumbar sacral segment. Nine cases were on the left foot and 12 cases were right foot. The ulcer area in out-below side of the fifth metatarsal bone was in 6 cases, in basement of the fifth metatarsal bone in 5 cases,in lateral of calcaneocuboid joint 2 cases, and in lateral of subtalar joint in 8 cases. Surgical procedure included plantar medial release technique, tendo-chilles lengthenning, and three-joint partial osteotomy. During operation,the skin and soft tissue of ulcer were resected and the incision was sutured when the ulcer was in the lateral of subtalar joint, and ankle Ilizarov external fixation for extension was installed after correcting talipes equinovarus partially. For the legacy skin ulcer in weight-bearing area,the Shenrg-ji cream (Chinese characters) was used after operation for one time per day until the wound healed. Adjusting Ilizarov external fixation for correcting residual deformity until it is satisfaction. During this process weight-bearing walking was keeping., Results: All cases were followed up from 6 to 52 months with an average of 28 months. All ulcers were healing and no recurrence. The ulcer healing time was 14 to 36 days postoperative (26 days in average). Eighteen deformity feet were corrected satisfactorily, and recovered fully plantar foot, 3 feet appeared mild deformity after 1 year. Ankle joint activity appeared limited in 15 cases and got completely rigidity in 6 cases. The AOFAS increased from preoperative 34.0±7.2 to 86.0±8.5 postoperatively; 8 feet got excellent, 10 good,3 fair., Conclusion: The clinical effect of Ilizarov technique combined with limited operation and the Sheng-ji cream in correction of the talipes equinovarus with skin ulcer in weigh-bearing area on account of neurotrophic malnutrition is satisfactory, the surgical method is simple and the treating course in security, and serious complications can be avoided.

At present, treatment for chronic radial head dislocation mainly focus on open reduction, and conservative treatment is invalid. While the surgical procedure generally applies to extend ulnar, short radial, reset radial head. Repair or reconstruction of annular ligament is the focus of study. For the dysfunction, radial head resection served as the final choice. Early diagnosis is the key to the treatment of chronic radial head dislocation. While ulnar or radial osteotomy and non-reconstruction of annular ligament is still the main trend of today.

Wolff law was an adaptable principle of bone, Tension-Stress Principle was equal to Distraction Osteogenesis or Distraction Tissue Regeneration, The Natural Reconstruction theory was a new orthopedic perspective proposed by Prof. QIN after deformity correction using Ilizarov technology. The thought about their relationship originated from a social phenomena, that the crowds and the confusion about export choice in Beijing's subway. Ilizarov technology and Wolff law were one concept related to Mechanics, and the former is completely in line with the latter. In other words, Ilizarov technology is an extension of Wolff law, is a repeated process of micro-trauma and continuous repair of bone trabecular initiated by moden engineering, just trabecular formed along the tension-stress direction. With adjustment of mechanical force,doctor can control the process of fracture healing and bone remolding to a certain extent. Natural Reconstruction theory enlarged the defined range of Wolff law obviously. Not only guided orthopedics clinical and basic research,but also related to the dialectical thinking of the doctor-patient relationship in sociology. There was an inevitable connection among Wolff law, Ilizarov technology and Natural Reconstruction theory. The history of discovery and understanding was a continuous process of thinking,practice and integration.

Objective: To study surgical strategies for ankle and foot deformities secondary to spina bifida and treatment methods for different types of deformities., Methods: From January 1990 and July 2009, 107 patients with ankle and foot deformities secondary to spina bifida were retrospectively analyzed. There were 44 males and 63 females, with an average age of 17.7 years (rangd from 1.3 to 52 years). Among the patients, 58 patients had double ankle deformities, 49 patients had unilateral deformities (22 cases on the left side, 27 cases on the right). Ninety-nine patients with equinus deformities were treated by achilles tendon lengthening and tendon transfering; 25 patients with talipes were treated by release of anterior tendon of ankle and tendon transfer; 17 patients with valgus and varus deformities were treated by tendon transfer and calcaneal osteotomy; 15 patients with flail deformities were managed treated by bone fusion between calcaneus and talus and shortening of achilles tendon; 9 patients with claw toe deformities were treated by bone fusion of interphalangeal joint or Ilizarovs distraction. AOFAS (American Orthopaedic Foot & Ankle Society) comprehensive scoring system was used to evaluate subjective pain and objective functional., Results: Seventy-nine (127 feet) of 107 patients were followed up, and the duration ranged from 48 to 180 months (averaged, 64 months). According to AOFAS scoring system, the results were rated as excellent in 89 feet, good in 26 feet, moderate in 9 feet and poor in 3 feet., Conclusion: Treatment strategies for ankle-foot deformities mainly included four principles, deformity correction, muscular balance, joint stability and reservation of foot elasticity. Different combined procedure was applied for different foot deformities and received good therapeutic effects.

Objective: To study therapeutic effects of limited internal fixation combined with external fixation in the treatment of deformity of distal end of femur, as well as to explore its effect on the function of knee joint., Methods: From June 2004 to June 2009, 38 patients with deformity of distal end of femur were treated with osteotomy at femoral supracondylar and internalfixation combined with hybrid external fixator. Among the patients, 19 patients were male and 19 patients were female, ranging in age from 14 to 58 years, with an average of 26 years. Six patients had genu valgum, 4 patients had varus of the distal end of femur, 2 patients had varus combined with rotation deformity,3 patients had genu recurvation and 23 patients had deformity of genuflex. The function of knee joint was evaluated according to Dimeglio clinical assessment after the treatment., Results: All the patients were followed up and the during ranged from 6 to 48 months, with a mean period of 23 months. All the deformity obtained satisfactory correction, without complications such as limitation of motion of the knee joint, under correction or over correction,displacement of the section of the osteotomy,internal fixation loosening and fracture. Nineteen patients got an excellent result, 12 good, 5 poor and 2 bad., Conclusion: Supracondylar femoral osteotomy and internal fixation combined with hybrid external fixator is a safe and effective surgery for the treatment of deformity of distal end of femur. This method combines the advantages of internal fixation and external fixation.

Objective: To analyze the incidence, clinical features, deformity categories and orthopedic treatment of foot and ankle deformities caused by spinal bifida., Methods: The charts of the patients received surgical treatment between January 1990 and July 2009 were studied retrospectively, and the data were analyzed., Results: One hundred and seven cases of foot and ankle deformities caused by spinal bifida received surgical treatment and were included. There were 44 male and 63 female patients. The average age was 17.7 years (range, 1.3 - 52.0 years). And 50.5% (54/107) of cases were over 18 years old and had spinal bifida occulta, and the other 49.5% had spinal bifida manifesta. There was only one case of thoracic spinal bifida (T(3-8)), while the other 106 cases had lumbosacral vertebrae cleft (mainly L(3) to Sacrum). Among a total of 165 feet, unilateral involvement was found in 49 cases (22 cases on the left side, 27 cases on the right side), bilateral involvement in 58 cases. Combined ankle-foot deformities included 76 varus talipes, 23 talipes valgus, 15 flail feet, and 51 other foot deformities. Other site deformities, as a result of spinal bifida, included knee flexion or hyperextension deformity in 4 cases, hip deformity (hip adduction, flexion, or hip dislocation, pelvic tilt, lower extremity discrepancy, etc.) in 17 cases, and urinatory dysfunction and defecation in 30 cases. Twenty-nine of 54 cases with spinal bifida occulta failed to be diagnosed in other hospitals and the misdiagnosed rate reached 53.7% (29/54). Corrective surgery was performed in only 26 patients. And 50.5% (54/107) of patients (over 18 years old) had severe foot and ankle deformities due to a failure of prior surgical treatment., Conclusions: Spinal bifida is the most commonly found in the lumbosacral vertebrae. Although the main pathogenesis is developmental abnormalities of spinal cord and nerve, the secondary deformity is usually located on the foot and ankle. Some young orthopedic surgeons may not have enough awareness and treatment experience about this disease due to over-specialty of the orthopaedics, so the delay of early diagnosis and treatment is often found and many severe foot and ankle deformities occur.

A Setosphaeria turcica gene encoding the catalytic subunit of calcineurin was cloned using degenerated primers corresponding to conserved domains of Ser/Thr protein phosphatases and its complete cDNA (GenBank accession No. EF 407562) was obtained with RACE method. It's validated single copied model by southern hybridization. Furthermore, the CNA inhibitor Cyclosporin A (CsA) exhibited potent antifungal activity against conidial germination and appressorium formation of S. turcica. The inhibition ratio was positively correlated to CsA concentration. However, appressorium formation was more sensitive than conidium germination to the inhibitor at the same concentration. It was suggested that CNA might play an important role in the pathogenicity of S. turcica.

Aim: To establish recombinant HEK293-16 cell strain expressing chimera receptor EpoR/LR-F3/HAb18GEF via site-specific integration expression system., Methods: The HAb18GEF gene fragment was amplified by PCR and digested with Sac I/Not I, and then cloned into the multiple clone site downstream of EpoR/LR-F3 gene of eukaryotic expression vector pCEL2f. The obtained pCEL2f/HAb18GEF vector, checked by partial nucleotide sequencing and restriction endonuclease digestion, was co-transfected with POG44 vector into HEK293-16 cells by mixing with Lipofect AMINE2000 reagent. After selection with hygromycin B for one month, transient and stable expression of EpoR/LR-F3/HAb18GEF were detected by indirect immunofluorescence staining, flow cytometry assay, and Zeocin test., Results: The recombinant pCEL2f/HAb18GEF vector, containing fused EpoR/LR-F3/HAb18GEF gene with correct open reading frame, was successfully constructed. It was confirmed that both EpoR and HAb18GEF were expressed efficiently on the membrane of co-transfeced HEK293-16 cells. 12 stably-expressed clones, sensitive to Zeocin, were finally obtained, which presented 99.93% EpoR-positive cells with strong immunofluorescence intensity of 1036.39, and simultaneously presented 99.51% HAb18GEF-positive cells with strong immunofluorescence intensity of 652.72., Conclusion: HEK293-16 cell strains stably expressing EpoR/LR-F3/HAb18GEF chimera receptor have been developed successfully, which lays foundation for MAPPIT (Mammalian protein-protein interaction trap) screening of binding molecule of hepatoma associated antigen HAb18G/CD147.

Objective: To explore the Ilizarov mini-invasive distraction technique for the treatment of severer ankle and foot deformities secondary to ischemic contracture of the leg., Methods: Based on the tension-stress low of Ilizarov, a serial of adjustable three dimensions external distractive instrument was developed in our department. From April 2002 to March 2004, 8 patients with ankle and foot deformities secondary to ischemic contracture of the leg induced by trauma and fracture were treated with the distractive instrument. Of them, 4 patient were male and 4 female, aged from 13 to 31 years with an average of 23 years. Five affected legs were in the left and 3 in the right. Preoperative abnormal style included talipes equines in 6 feet and equinovarus in 2 feet, with extensive scar contracture in the legs. Five patients suffered from failure of soft tissue release before, two patients with severe bony deformity of the feet were underwent limited foot triple osteotomy in this department before the distractive correction. The distraction was begun from 7 d after operation and distractive time from 29 to 60 d with an average 46 d., Results: All of the 8 patients were followed up from 10 months to 29 months, with an average of 13 months. All of deformities in the feet were corrected satisfactorily, full feet contacted with the ground in stand or walking and achieved with good function. No complication, such as pin tract infection, skin necrosis and neurovascular injury was occurred in this group., Conclusions: Mini-invasive distraction technique for treatment of severe ankle and foot deformity secondary to ischemic contracture of the leg is safe and mini-injury, it is also an effective approach for the treatment of various kinds of rigid foot anomaly.

Aim: To screen human anti-gamma-sm (gamma-seminoprotein) light chain (Lc) with guided selection of murine Fd fragment., Methods: The human Lc repertorie genes were amplified by RT-PCR from PBMC in patients with prostate cancer, and cloned into the phagemid vector pComb3X with murine Fd gene against gamma-seminoprotein to construct the human-mouse hybrid Fab antibody library. The size of the library, antibody gene recombinant percentage and diversity were identified by colony counting, plasmid digestion and colony sequence analysis, respectively. Purified gamma-sm was used as antigen to screen the displayed phage hybrid antibody library rescued by helper phage M13K07 for three rounds. The positive clones were selected by ELISA with pIII-fusion antibody and then the sequences of light gene in the positive clones were analyzed by IMGT-VQUEST., Results: A 1.2x10(7) CPU human-mouse Fab antibody library was constructed with 90% Lc gene recombinant and great diversity. After 3 rounds' panning with gamma-sm, 5 positive clones were selected by ELISA and 2 clones with higher affinity were selected. Sequence analysis suggested these two positive clones contained the same light gene with high V(L) homology to human germline gene IGKV4-1*01., Conclusion: Human anti-gamma-sm light chain was successfully screened by constructing mouse-human hybrid Fab phage antibody library with murine Fd-guided selection.

Objective: To shorten the time of external skeletal fixation on legs, and enhance quality of limb lengthening, avoid complications of shortening, bending, twisting and etc., Methods: Insert pin transcortical to attack external skeletal fixation simultaneously, put un-reaming locked intramedullary nail (do not insert distal locked screw) into endosteum of lengthening bone. After the legs achieved predetermined length, insert distal locked screw and then remove external skeletal fixation, locked intramedullary nail, then maintain consolidation of rehabilitation., Results: The group lengthened legs for 412 cases. The range of lengthening was 3 to 18 cm. Mean length was 7.6 cm. The mean time for needed external skeletal fixation was 20 d/cm. The mean time of osteogenesis was 56 d/cm. For complications, there were 3 tibias ununion cases and 1 varus ankle. All cases were treated undergoing twice., Conclusions: The method reduces the time for needed external skeletal fixation visibly, enhances the quality of limb lengthening remarkably, prevents complications of shortening new bone, deformity, bending and re-fracture which do not effect the healing time. This is a new choice of limb lengthening.

Aim: To compare characteristics of polyclonal anti-sera against extracellular domain of hepatoma-associated antigen HAb18G/CD147(HAb18GEF) generated by different immunization schemes., Methods: BALB/c mice were immunized with GST-HAb18GEF fusion protein expressed in E.coli (routine immunization method), recombinant eukaryotic expression plasmid pcDNA3/HAb18G (intramuscular injection) and pcDNA3/HAb18G plasmid followed by human hepatoma cells booster (DNA-cell booster), respectively. The titers and Ig subclasses of polyclonal anti-sera against denatured and natural HAb18GEF were detected by indirect ELISA and cell ELISA, respectively. The specific binding of polyclonal anti-sera prepared by different immunization schemes to denatured HAb18GEF was analyzed by Western blot. The specific binding of polyclonal anti-sera produced by DNA-cell booster immunization to natural HAb18G antigen on hepatoma cells was detected by immunofluorescence staining., Results: GST-HAb18GEF immunization could induce polycolonal antibody IgG1 with higher titer mainly against denatured or linear epitopes on HAb18GEF. Antibody induced by pcDNA3/HAb18G intramuscular immunization was IgG2a with lower titer against natural epitopes on HAb18GEF. DNA-cell booster immunization could induce generation of polycolonal antibody IgG2a and IgG1 of moderate titers against the native epitopes on HAb18G., Conclusion: The polycolonal sera with different titers against different epitopes on HAb18GEF can be induced by different immunization schemes.

Objective: To study the role of the synchronized springy lengthening apparatus for the tibia and calcaneal tendon designed by the author in preventing the clubfoot of secondary to the Ilizarov tibia lengthening., Methods: Based on the Ilizarov tibia lengthening apparatus, a special synchronized springy lengthening apparatus for the tibia and calcaneal tendon was designed. The tibial was made of distal and proximal 2 rings respectively and 4 threaded rods, and the calcaneal was made of a half ring, 2 hinges and a threaded rod with spring. The half ring was fixed to the calcaneus by 2 crossed wires. The fracture tibia and fibula, ankle joint, talocalcaneal joint were attached to the apparatus. At the same time of tibia lengthening, the soft tissue was simultaneously stretched, the ankle joint could move, and the leg could bear weight. If the clubfoot angle was larger, the percutaneous fasciotomy of calcaneal tendon was performed; if the angle was less than 20 degrees, the pes deformities were corrected only by the stretch of calcaneal tendon., Results: Seventy-seven patients' tibia were lengthened averagely 4.6 cm, with an average speed of 0.7 mm/d. The healing made tibia lengthened, and the index was 1.35 months/cm. There were not the secondary varus and valgus deformities and clubfoot in all the patients. The clubfoot with 100-400 angle of the 16 patients were corrected after tibia lengthening., Conclusions: The new apparatus coincides with the biomechanical principle and can effectively prevent the secondary deformities of foot such as clubfoot, talipes varus and valgus after tibia lengthening procedure.

Objective: To study the methods and effects of Ilizarov distraction technique in treating the flexion deformity of the knee of arthrogryposis multiplex congenita., Methods: Between August 1998 and February 2003, 6 patients (10 knees) with the arthrogryposis multiplex congenita were treated, 4 patients in double knees, 5 males, 1 female, mean age 8 years and 2 months, ranged from 3 years and 7 months to 13 years. The preoperative flexion degree was averagely 51 degrees. The patients accompanied 13 other parts malformation of limbs. The modified Ilizarov distraction apparatus of the knee was used. While installing the apparatus in the operation, the knees should be kept in the of location of maximum extension, the center of joint hinges on the apparatus should be placed towards the rotatory center of the knee, two groups of 2 mm K-wires were passed through the femur and tibia around the knee, who were fixed on the proximal and distal rings. Distraction was started after the surgical procedure 5 days via rotating the threaded rods at the posterior of the knee, at an average of 2 to 3 mm per day, at the first week, after 2 weeks with the rate modified to 2 mm per day, up to the knee extended to 0 degrees. The accompanied deformities of the hip and/or the foot might be corrected at the same time or next time. The average duration of the distraction was 37 days (23-48 days). During the correction all limbs might undergo weight. After 2 weeks at the end of distraction the fixator was removed and the patients could walk by a long-leg brace., Results: Ten knees with the flexion contracture were sufficiently corrected without severe complications. Nine knees of all were followed up at an average time of 1 years and 3 mouths, no recurrence of the deformity was seen in all patients, their function of walk was significantly improved., Conclusions: Ilizarov technique is a simply, safe and effective method for managing the flexion deformity of the knee of the arthrogryposis multiplex congentia. The procedure is conformable to the biological theories and microsurgical principles.

To express the extracellular fragement of hepatoma associated antigen HAbl8G(HAb18GEF) in E. coli efficiently in a non-fusing way, the cDNA of HAb18GEF gene was inserted into prokaryotic expression vector pET21a + . The secondary structure and codon adaptation of translational initiation region (TIR, from-30 to + 39) in mRNA of recombinant vector HAb18GEF/ pET21a + was predicted simultaneously by computer-aided design. Stable Stem-Loop structures and many low-usage codons were detected in mRNA-TIR of non-optimized recombinant HAb18GEF/pET21a + vector. The stability of mRNA-TIR in recombinant HAb18GEF/pET21a + vector was reduced with following methods: (1) optimization of secondary structure (2) optimization of codon adaptation. These optimization were realized by non-continual site-directed mutagenesis without changing any amino acid sequence in TIR. After being checked through restriction endonuclease digestion and confirmed through nucleotide sequencing, the pre-optimized and post-optimized recombinant vectors were transformed into competent E. coli JM109-DE3. The resulted recombinant clones were selected randomly and induced by IPTG at 37 degrees C. The induced production of these recombinants was analyzed by SDS-PAGE, indirect ELISA, Western blot, and cell fractionation assay. The amount of HAb18GEF mRNA was also detected by RNA dot blot between pre-optimized recombinant and post-optimized recombinant. The results revealed that recombinant non-fused vectors HAb18GEF/pET21a + were successfully constructed and optimized in the secondary structure and codon adaptation of TIR respectively. The HAb18GEF was expressed efficiently in a non-fusing way in recombinant E. coli by secondary structure optimization or codon adaptation optimization. Whereas, no expression of HAb18GEF was detected in pre-optimized recombinants. The non-fused expression products-HAb18GEF, mainly as inclusion body in E. coli, yielded highly above 29.3%. A trait of expression HAb18GEF was also detected both in intermembrane space and in culture medium due to over-expression and cell leakage. Difference in non-fused expression level of HAb18GEF between secondary structure optimization and codon adaptation optimization was negligible. No difference in amount of transcribed mRNA of HAb18GEF between the pre-optimized and the post-optimized recombinants was detected. To sum up, it's feasible to express hepatoma associated antigen HAb18GEF in a non-fusing way by reducing the stability of TIR in mRNA.

Aim: To construct a anti-dodecane-tertraacetic acid-yttrium(DOTA-Y) immune Fab phage antibody library., Methods: BALB/c were immunized with BSA-Y-DOTA which was prepared by DOTA-conjugated BSA and chelated with Y. After determination of anti-serum, total RNA was extracted from splenic lymphocytes of immuned mice. The heavy chain Fd and light chain Kappa genes repertoires of immunoglobulin were amplified respectively by RT-PCR, and then the amplified products were cloned into the reconstructive phage vector pComb3M to construct anti-DOTA-Y Fab antibody. And then, the recombination rate, diversity and display of Fab antibody library were identified by restriction endonuclease digestion, DNA sequencing and ELISA., Results: BSA-Y-DOTA was prepared successfully, and a higher titer of immune sera was achieved. The amplified gene fragments of Fd and Kappa chain by RT-PCR were correct and the length was with about 650 bp, and were inserted exactly. The sink size of Fab phage display library reached 8 x 10(7), the re-combination rate was about 90%, and it possesed great diversity. In addition, ELISA detection showed that there was Fab expression on the phage library., Conclusion: An immune Fab phage antibody library of DOTA-Y has been constructed successfully, which lays a solid foundation for screening specific anti-DOTA-Y antibody.

Objectives: To explore the effect of IL-18 on peripheral blood monocytes (PBMCs) from chronic hepatitis B (CHB) patients and HBV DNA released by HepG2.2.15 cells, which were transfected with the gene of HBV., Methods: PBMCs were isolated from 25 healthy persons and 25 CHB patients, which were co-cultured with HBcAg and IL-18 at different concentrations for 72 hours. The level of IFN-gamma in the culture supernatant of PBMCs was determined by ELISA. One patient' PBMCs were co-cultured for 96 hours with various concentrations of IL-18 and HepG2.2.15 cells which had been cultured for 24 hours, the supernatant was collected to detect HBV DNA level by PCR., Results: When PBMCs were stimulated by HBcAg and IL-18 at various concentrations, the levels of supernatant IFN-gamma in the CHB group were much higher than those in the normal control group (at 0.2ng/ml: t=11.7, P<0.01; at 1.0ng/ml: t=16.19, P<0.01; at 5.0ng/ml: t=20.12, P<0.01), especially when the PBMCs were stimulated by HBcAg, IL-18 and IL-12 (1313.20pg/ml+-187.76pg/ml vs. 390.75pg/ml+-43.23pg/ml, t=23.94, P<0.01). The IFN-gamma level in the patients who were stimulated by HBcAg alone was much lower than the levels in the patients who were stimulated by HBcAg and IL-18 at various concentrations, and which were lower than those in the patients stimulated by HBcAg, IL-12 and IL-18 at the same concentrations (light: t=2.2, P<0.05; moderate: t=2.97, P<0.05). The HBV DNA content in the supernatant of co-cultivation with HepG2.2.15 cells and PBMCs was much higher than that of the two kinds of cells stimulated by HBcAg and IL-18 at various concentrations or HBcAg, IL-18 and IL-12/IFN-a1b., Conclusion: IL-18 can improve the PBMCs from CHB patients to produce a great deal of IFN-gamma, so it has a good application prospect in two aspects: immunoregulatory effects and increasing the ability to kill the cells infected with virus.