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Objective To construct eukaryotic expression recombinant plasmid containing human granulysin(GLS) and investigate the effect of GLS expression in macrophage RAW264.7 cells on the bactericidal activity against intracellular Mycobacterium tuberculosis.Methods GLS gene was amplified by nested-polymerase chain reaction(PCR) from human cytotoxicity T lymphocyte(CTL) activated by allogenic antigen,and inserted into pBudCE4.1 vector to construct recombinant plasmid.Subsequently,the plasmid was transfccted into RAW264.7 cells which were infected with Mycobacterium tuberculosis H37Rv.The expression of GLS was detected by nested-PCR and immunocytochemistry method.The RAW264.7 cells were lysed after transfected for 96 h,then acidfast stained,cultivated and colony count were done to determine the intraeellular bactericidal activity of GLS.The data were analyzed by t or t' test.Results The pBudCE4.1/GLS eukaryotic expression recombinant plasmid was successfully constructed.The transcriptional and translational expressions of target gene GLS were detected in RAW264.7 cells which were infected with Mycobacterium tuberculosis H37Rv.The bacterial load in macrophages of phorbol myristate acetate(PMA)+pBudCE4.1/GLS group,PMA+pBudCEA.1 group and non-activated group were 1.44±1.25,3.16±0.20 and 3.59±0.21,respectively.The differences between groups were all significant (t=2.403,t=2.854,both P＜0.05).Conclusion Eukaryotic expression recombinant plasmid carrying human GLS gene expressed in macrophages has strong bactericidal activity against intracellular mycobacteria,which provide information for the further study on therapeutic vaccine against Mycobacterium tuberculosis.
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Objective; To clone, sequence and analyze the coding sequences of the Mr 15000 and Mr 9000 active segments of natural granulysin derived from human CTLs activated by allogenic antigen ,in order to establish the basis for further purifying and investigating the immune - impairing mechanism of granulysin. Methods; The coding sequences of the Mr 15000 and Mr 9000 granulysin gene were amplified from the total RNA of activated CTLs of healthy person after reverse transcription by Nested - PCR, inserted into pET32a ( + ) vectors and then transformed into E. Coli TOP10, respectively. The recons were identified by PCR, endonuclease digestion and sequencing. Results: The whole coding sequences of the Mr 15000 and Mr 9000 active segments were successfully cloned as expected. The accurate pET32a - Mr 15000 and pET32a - Mr 9000 recons were obtained through Colony - PCR, endonuclease digestion and sequencing. There lied polymorphism on the 119 th amino acid of the product encoded by GLS gene. Conclusion; The coding sequences of the Mr 15000 and Mr 9000 active segments of human granulysin can be obtained and cloned by the methods mentioned above and can be used for subsequent research.
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Objective To characterize the effects of TPP1 knockdown on Pot1a and Pot1b localization at telomeres and on the telomere end protection.Methods Knockdown of endogenous TPP1 in mouse embryonic fibroblasts(MEFs) with the retrovirus vector encoding shRNA targeting TPP1,IF/PNA-FISH was performed to determine the localization of Pot1a and Pot1b at telomeres,and TdT-FITC was applied to characterize the effects on the function of telomere end protection,cellular senescence was analyzed by SA-beta gal staining,and phosphorylated p53ser18 and p21 were examined by Western blotting.Results Pot1a and Pot1b were unable to localize at telomeres in about 65% of MEFs with TPP1 knockdown,while that was found in less than 5% of MEFs without TPP1 knockdown(t=10.96,P
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Objective To study the route of intrauterine infection of chlamydia trachomatis (CT) Methods Seven hundred and seventy two cervical samples from in women and 105 matched maternal labom neonatal samples composed of cervical samples,cord blood, amniotic fluid, conjunctival and nasopharyngeal samples of neonate were detected by PCR SSCP and DNA sequencing technique Results CT were detected in 87 of 772 (11 3%) cervical samples In the 81 matched maternal infant samples from pregnant women with cervical CT positive, CT were not detected in all of the cord blood samples In the 30 CT positive neonatal samples, 26 were from cases of vaginal delivery and 4 from cases of caesarean section Statistical analysis showed a significant difference between the groups of caesarean section and the vaginal delivery ( P
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Objective To investigate the relationship between auxotypes,antibiotic resistance and plasmid of Neisseria gonorrhoeae. Methods 83 isolates of Neisseria gonorrhoeae from Chongqing during the period from January 1994 to November 1995 were examined.Results Eight auxotypes were detected in 83 strains. The prevalent auxotypes were proline requiring strains (51.8%) and nonrequiring (18.1%). The agar dilution method was performed to detect the susceptibility of 83 isolates to pencillin,tetracycline and spectinomycin, the results indicated that 15.6% of strains was penicillin resistant,70% was tetracycline resistant, 1.2% was spectinomycin resistant. Plasmid profile of 44 isolates,which was tested by rapid alkaline extraction method, showed that 70% of the strains carried 24.5 Md conjugative plasmid, 80% carried 2.6Md cryptic plasmid, 66% carried both 24.5Md and 2.6Md plasmids, and no plasmid was found in 16% of the strains. In Pro - strains both penicillin resistant strains and 24.5Md plasmid carrying strains were more common than other auxotypes (P
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30mg partially purified pBMP was implanted into right quardriceps muscle in each rabbit. Whole blood was collected from ear veins before and after the operatin. Lymphocyte proliferation response to nonspecific mitogen PHA and to specific pBMP were measured by lymphocyte transformation test in vitro, using H—labelled thymidine incorporatcon method (~3H—TdR). The skin test and histological investigation of pBMP were also performed. It has been shown that no significant changes of cellular immunity in rabbit were observed after pBMP implantation; the immunogenicity of pBMP was weak. The likelihood of chinical use of pBMP is provided.
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This study re-evaluated the application of Gram's stain in diagnosing gonorrhea in women by comparing with gonococci culture.615 samples from 500 cases in clinic of Gynecology were studied.The results showed that sensitivity, specificity,positive predicative value, negative predicative value were 94.9% 95.2% ,74%,99.2% respectively.It is suggested that Gram's stain may be used in screening healthy subjects a-nd in diagnosing gonorrhea in women.