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袁作为,帅光泽,张君,胡接力,郑建.HIV跨膜蛋白gp41和gp36原核表达及抗原性研究[J].陆军军医大学学报(原第三军医大学学报),2010,32(21):2281-2285.
Yuan Zuowei,Shuai Guangze,Zhang Jun,Hu Jieli,Zheng Jian.Expression of HIV trans-membrane protein gp41 and pronucleus gp36 and their antigenicity[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(21):2281-2285.
袁作为,帅光泽,张君,胡接力,郑建.HIV跨膜蛋白gp41和gp36原核表达及抗原性研究[J].陆军军医大学学报(原第三军医大学学报),2010,32(21):2281-2285. DOI:
Yuan Zuowei,Shuai Guangze,Zhang Jun,Hu Jieli,Zheng Jian.Expression of HIV trans-membrane protein gp41 and pronucleus gp36 and their antigenicity[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(21):2281-2285. DOI:
目的 构建HIV跨膜蛋白gp41和gp36原核表达载体,诱导表达HIV抗原,为HIV快速诊断和蛋白疫苗研究提供材料基础。 方法 用PCR方法对HIV-1包膜蛋白gp41和HIV-2包膜蛋白gp36基因序列进行全长、截短扩增,分别或以融合表达方式克隆入原核表达载体pQE30、pET32a(+)及pGST-MOLUC,重组质粒经异丙基硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside
IPTG)诱导表达,并经Ni-NAT或谷胱甘肽-Sepharose-4B层析柱亲和纯化HIV跨膜糖蛋白抗原,对获得的纯化抗原进行Western blot检测,利用HIV抗原制备ELISA检测试剂盒,并分析HIV临床样本。 结果 成功构建了HIV包膜蛋白的原核表达载体,利用亲和层析纯化得到了纯度较高的HIV包膜蛋白;Western blot分析结果证实表达纯化的HIV跨膜蛋白gp41(aa.538~718)、gp36(aa.533~764)以及gp41-gp36均能和HIV阳性血清反应;ELISA试剂盒检测临床样本显示,特异性达95%以上,灵敏度达100%。 结论 获得了有生物学活性的HIV截短包膜蛋白,并可用于HIV快速检测试剂盒的研发。
Objective To construct the recombinant plasmids with HIV-1 gp41 and HIV-2 gp36 genes and induce the expression of HIV antigens for the rapid diagnosis of HIV and development of protein vaccine. Methods Full-length genes and/or fragment genes encoding HIV-1 trans-membrane protein gp41 and HIV-2 trans-membrane protein gp36 were amplified by PCR. Target genes were inserted into the expression vectors pQE30
pET32a(+) and pGST-MOLUC
respectively. Recombinant vectors were transformed into E.coli BL21 and induced by IPTG. Exp
ressed fusion proteins were purified by Ni
2+
-affinity chromatography or GST-affinity chromatography. Antigenicity of purified proteins was identified by Western blotting and ELISA were prepared using the purified proteins. HIV samples were analyzed. Results The pronucleus vector of HIV trans-membrane protein was successfully constructed. HIV transmembrane protein with a rather high purity was obtained by Ni
2+
-affinity chromatography or GST-affinity chromatography. Western blotting showed that the expressed products including gp41 (aa.538-718)
gp36 (aa.533-764) and gp41-gp36 reacted to positive HIV in serum. HIV-1
+
or HIV
-
were detected with ELISA in HIV samples with a sensitivity of 100% and a specificity of over 95%. Conclusion The biological activity of HIV truncation cross membrane proteins can be used in research and development of HIV rapid diagnosis kits.
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