- ZHAO Yueran,YOU Li,GAO Chunyi et al( Institute of Basic Medicine, Shandong Academy of Medical Sciences , Shandong Cancer Biotherapy Center, Jinan 250062)
Objective To express recombinant human IL - 10 in E. coli. Methods A hIL - 10 DNA fragment, with a length of about 500bp, was amplified from the RNA of leukemia cell strain K562 by RT - PCR and cloned to plasmid pSK( + ) ,and the cloned DNA fragment was sequenced. The recombinant plas-mid pSK/IL- 10 was digested with EcoRI and BamHI, then hIL- 10 fragment was isolated and inserted to the corresponding restriction site on procaryotic expression vector pBV220.The recombinant plasmid PBV/IL - 10 was identified by enzymogram and transformed to E. coli, then expressed by induction at 42℃ .The expressed product was identified by SDS-PAGE and Western blot and renaturalized with glutathione buffer, and the inhibitory effect of it on the production of IFN - γ in PBMC was detected by RT - PCR. Results The length of DNA fragment amplified by RT - PCR was consistent with that of hIL - 10 cDNA. DNA sequencing of pSK/ IL- 10 revealed that the cloned DNA sequence was identical to that of reported hIL- 10 cDNA.SDS - PAGE proved that the expressed product, with a relative molecular weight of 18000,contained about 20% of total somatic protein. Western blot showed that the recombinant protein could specifically bind to anti-hIL-10 antibody and,after being renaturalized, could inhibit the production of IFN-γ in PBMC significantly. Conclusion A recombinant bacterial strain for expressing hIL - 10 with biological activity was successfully constructed.
2002 06 [Abstract][OnlineView][Download 584k] - SI Shaoyan, ZHENG Yanhua, LI Xinyuan et al( Department of Laboratory Medicine, 306th Hospital of PLA, Beijing 100101)
Objective To express and purify recombinant human Tau protein. Methods The recom-binant bacterial strain containing Tau gene was induced with IPTG, and the expressed product was purified by sonication, salting out with ammonium sulfate, ion exchange charomatography and electric elution, then purified by SDS - PAGE, non - denaturing PAGE, Western blot and amino acid sequencing of N - terminal. Results The recovery rate of purified Tau protein was 28.5% . Either SDS - PAGE or non - denaturing PAGE profile showed a single protein band with a relative molecular weight of 59000. The sequence of 5 amino acids at the N - terminal of the protein was NH2 - Met - Ala - Glu - Pro - Arg - . Conclusion The methods in this paper were effective in the purification of recombinant human Tau protein. It laid a foundation of study on pathogene-sis and early diagnosis of Alzheimer's disease.
2002 06 [Abstract][OnlineView][Download 473k] - SHI Zhilin,LI Tiyuan,DU Hong et al( Shenzhen People' s Hospital, Medical College, Jinan University, Shenzhen 518020)
Objective To develop recombinant human pancreatic kallikrein and lay a foundation of gene thereapy of hypertension. Methods Synthesize human pancreas cDNA by RT - PCR, amplify kallikrein gene, digest with Xho I and EcoR I and insert into the pET- 28b( + ) vector. The clones were identified by enzyme digestion and sequenced,and fusion protein was expressed under the induction of IPTG.Results SDS - PAGE profile showed a clear protein band with a relative molecular weight of 31800. Western blot proved that the expressed product showed specific antigenicity to human serum kallikrein. Conclusion Human pancreatic kallikrein gene was successfully cloned and expressed. It laid a foundation of further development of recombinant kallikrein and gene therapy of hypertension.
2002 06 [Abstract][OnlineView][Download 476k] - JIN Hongtao, JIN Ningyi, WANG Hongwei et al (Institute of Virology, Quartermaster University of PLA, Changchun 130062)
Objective To express anti - HIV - 1 gp120 ScFv gene in E. coli, purify expressed product and detect the biological activity of it. Methods The anti-HIV - 1 gp120 ScFv gene was inserted into vector pET28, then transformed to E. coli for expression. The expressed product was degenerated and renatu-ralized, and purified by Ni - NTA affinity chromatography. The purified ScFv was identified by reaction with standard gp120 antigen. Results The expressed product existed mainly in the forms of inclusion bodies and contained 51% of total protein. After being purified by Ni - NTA chromatography, the purity of it reached above 85% . The purified ScFv recognized standard gp120 antigen specifically. Conclusion ScFv with biological activity was highly expressed in E. coli.
2002 06 [Abstract][OnlineView][Download 493k] - LI Taiyuan , JIN Ningyi, DING Zhuang et al( Department of Veterinary Medicine, Agriculture College, Yanbi-an University, Longjing 133400)
Objective To express HN gene of Newcastle disease virus(NDV) strain F48E8 in sf- 9 insect cell by a novel baculvirus system(Bac to Bac) .Methods A recombinant transposable vector pFast HN was constructed by inserting HN gene into transposable vector pFast Bac I and transformed into competent cell DN10,then the goal gene was transposed into shuttle vector Bacmid.The recombinant Bacmid HN was screened and transfected to insect cell sf - 9 for expression. Results Both SDS - PAGE and Western blot profiles showed a specific protein band with a relative molecular weight of 63000. Conclusion The HN gene of NDV strain F48E8 could be expressed in insect cell sf- 9 by a novel baculovirus expression system,and the expressed product contained about 10% of total cellular protein.
2002 06 [Abstract][OnlineView][Download 383k] - LI Shumin, LI Junzhi, WU Guangmou et al ( The Military Veterinary Institute, Quartermaster University of PLA, Changchun 130062)
Objective To observe the curative effect of LHRH - PE40 on ascitic tumor and solid tumor in BALB/c mouse model and provide a basis for the determination of dosages in pharmacological and toxi-cological tests.Methods Inoculate i. d. SP 2/0 cells into BALB/c mice, and inject with LHRH - PE40 4 days after inoculation, once a day for 8 days, using physiological saline as control. Meanwhile, inoculate s. c. SP 2/0 cells into BALB/c mice, and inject with LHRH - PE40 9 days after inoculation, every other day for 8 times, using physiological saline as control. Kill the mice 5 days after the last injection and observe the ascite and the growth of tumors in abdominal cavity. Weigh the tumors and calculate tumor inhibition rate. Results No ascite or tumors was observed in the mice inoculated i. d. with SP 2/0 cells. The tumor inhibition rate of the mice inoculated s. c. with SP 2/0 cells was 54% . Conclusion LHRH - PE40 can inhibit the growth of solid tumors in tumor-bearing mice.
2002 06 [Abstract][OnlineView][Download 543k] - WU Xueqiong, LI Hongmin, SHI Yingchang et al ( Tuberculosis Research Center, The 309 th Hospital of PLA , Beijing 100091)
Objective To study the curative effect of 3 kinds of tuberculosis DNA vaccines, i.e. MPT64, ESAT6 and Ag85A vaccines in mice. Methods Inject i. d. BALB/c mice with M. tuberculosis H37Rv for 8 weeks, divided the mice into 8 groups, and treat with physiological saline (A) , vector plasmid (B), BCG (C), M. vaccae (D), MPT64 DNA vaccine (E), ESAT6 DNA vaccine (F), Ag85A DNA vaccine and combined DNA vaccine ( H) respectively. Detect MPT64 - , ESAT6 - and Ag85A - serum antibodies by ELISA. The lungs, livers and spleens of the mice were isolated 2 and 5 months after treatment, weighed and subjected to pathological examination, bacterial colony counting, microscopy of lung tissue smear, and splenic lymphocyte transformation test. Results Antigen - specific antibody levels increased in all the mice immunized with DNA vaccine. No significant difference was observed in the splenic lymphocyte transformation rates of various groups 2 months after treatment. However, the bacterial numbers observed by microscopy of lung tissue smears, as well as colony counts in lung of various groups, decreased at different extents. The colony counts in lungs of group D and spleens of group E were significantly lower than control. Slightly pathological lesions were observed in the lungs of groups C, E and B. All the splenic lymphocyte transformation rates of the mice immunized with DNA vaccines increased significantly 5 months after treatment, and no significant difference was observed in the bacterial numbers in lungs and spleens of various groups. No significantly pathological change was observed in the lungs of group G or D. Conclusion The DNA vaccines showed curative effect on tuberculosis.
2002 06 [Abstract][OnlineView][Download 523k] - HAO Guorong, LI Qunying, YANG Lihua et al ( Changchun Institute of Biological Products, Changchun 130062)
Objective To deliver tetanus toxoid by a single administration using biodegradable micro-spheres . Methods Two kinds of biodegradable microspheres were prepared by the encapsulation of tetanus toxoid with 2 kinds of polylactide-co-glycolide (PLGA) polymers respectively using solvent evaporation technique. The surface morphology and sizes of the microspheres were observed by electron microscopy, and the antigen content and integrity were detected by Bradford method and SDS - PAGE respectively. The antibody response to TT was observed in guinea pigs. Results The microspheres were smooth and even in size. The mean diameters of the 2 kinds of microspheres were 8. 4μm and 9. 7μm, and the encapsulation rates of them were 48% and 56% respectively. No significant change was observed in the TT antigen integrity after encapsulation. Animal test showed that the anti - TT - IgG and neutralizing antibody titers induced by a single dose of TT microspheres were not significantly different from those induced by 3 doses of aluminium - adsorbed TT. The binding capacity of sera immunized with TT microspheres was also similar to that immunized with aluminium - adsorbed TT. However, the anamneotic response level induced by TT microspheres was significandy higher than that induced by aluminium - adsorbed TT. Conclusion A single dose of TT delivered by biodegradable microspheres induced continuous and high immune response level.
2002 06 [Abstract][OnlineView][Download 654k] - MENG Fanbo , YANG Ping, XIN Jihua et al ( China - Japan Union Hospital of Jilin University, Changchun 130031)
Objective To explore the effect of recombinant human hormone on action potential of ventricular muscle cell and calcium channel. Methods Study the action potential and influx calcium ion current of ventricular muscle cells of guinea pigs by using Langendorff model and patch - clamp technique. Results Ten minutes after the administration of recombinant human growth hormone, the plateau of action potential prolonged significantly. The APD50 and APD90 increased from 261. 14 ± 15.42 and 311. 23 ± 13. 7ms to 354.65 ± 31.22 and 401.33 ± 18.13ms respectively. The peak calcium ion current increased by about 40% , from 2.03 ± 0.09 to 2.85 ± 0.11 PA. Conclusion Recombinant human growth hormone prolonged the plateau of action potential and promoted the opening of calcium channel.
2002 06 [Abstract][OnlineView][Download 174k] - LI Kexi , TAN Ningzhi, LIU Yuqing et al ( Chengdu Institute of Biological Products, Chengdu 610063)
Objective To prepare and study the immunogenicity of pneomococcus type 6B capsular polysaccharide (6B - PNCPS) - tetanus toxoid (TT) conjugate vaccine. Methods 6B - PNCPS - IT was activated by Cyangen Bromide (CNBr) and reacted with adipic acid dihydrazide (ADH), then 6B - PNCPS - TT conjugate was prepared by carbodiimide - mediated coupling of 6B-PNCPS with tetanus toxoid (TT) . NIH mice were immunized with 6B - PNCPS - TT conjugate, using pure 6B - PNCPS as control, and the PNCPS antibody titers in the sera of them were detected by ELISA. Results Gel chromatography proved that, compared with that of 6B - PNCPS, the relative molecular weight of 6B - PNCPS - TT was large. The ratio of polysaccharide to protein was 1.42- 1.66. The conjugate vaccine showed the serological characteristic of 6B -PNCPS, and the liters of specific IgG induced by it was significantly higher than that induced by 6B - PNCPS. Conclusion The prepared 6B - PNCPS - TT conjugate vaccine showed good immunogenicity.
2002 06 [Abstract][OnlineView][Download 246k] - XU Chunxiao, YAO Lihong, ZU Dong et al ( State Key Laboratory of Molecular Virology and Genetic Engineering, Institute of Virology, Beijing 100052)
Objective To purify soluble tumor necrosis factor receptor Ⅱ from E. coli and prepare the receptor protein neutralizing TNF activity. Methods The recombinant protein was extracted by metal chelate Sepharose chromatography from E . coli after sonication and heat - treatment and analyzed for physical, chemical and biological properties. Results The recombinant protein reached a purity of above 95% . It can neutralize the bioactivity of TNF and inhibit the killing effect on cells. Conclusion TNF receptor protein was simply and effectively purified by the procedure. It laid a foundation of further study on TNF receptor.
2002 06 [Abstract][OnlineView][Download 632k]
2002 06 [Abstract][OnlineView][Download 68k] - LI Guangpu.LIu Jingye, WANG Pengfu et al( Changchun Institute of Biological Products , Changchun 130062)
Objective To prepare a protective addivive of freeze-dried live attenuated hepatitis A vaccine . Methods Live attenuated hepatitis A vaccine was prepared by culture of HA virus L - A - 1 strain in 2BX cells and lyophilized after addition of various protective additives. Results The protective additive composed of sorbite, sucrose, etc. showed good protective effect on live attenuated HA vaccine. The infectious liters of the vaccine decreased by not more than 1.0 Log CCID50 /ml after lyophilization. The result meets the Chinese Requirements for Biologies. Conclusion The protective additive composed of sorbite, sucrose, etc. was safe and effective, and suitable for the large - scale production.
2002 06 [Abstract][OnlineView][Download 127k] - YAN Zihou ,RONG Zuyuan.HE Ju et al( Chengdu Institute of Biological Products , Chengdu 610063)
Objective To establish a method for studying the antitumor effect of therapeutic BCG in mice. Methods Inject s. c. the therapeutic BCG at different dosages into the mice with solid tumor sarcoma 180(S180) or Ehrlich carcinoma(EC) and observe the inhibitory effect on tumors. Results BCG can signifi-cantly reduce the tumor weight of the mice with S180 .The inhibitory rate to tumor weight was above 30% . BCG can also prolong the survival time of mice with EC. The life prolonging rate was above 75 % . The therapeutic BCG at dosages of 12.5 and 6.25ng/kg showed good curative effect. Conclusion The method can be used for the antitumor pharmaceutical test of BCG in animals. .
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