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目的探讨人类嗜T淋巴细胞病毒(Human T-cell Lymphotropic Virus,HTLV)I型重组env抗原的表达。方法分析和选择HTLV-I env目的基因,将目的基因片段分别克隆入原核表达载体pQE80L,PCR和酶切鉴定重组子,诱导并亲和层析纯化表达重组env蛋白抗原,Western-blot检测重组env蛋白抗原活性,ELISA方法测试重组env蛋白抗原的特异性。结果 PCR扩增和酶切筛选得到阳性重组子pQE80L-env。SDS-PAGE显示重组蛋白相对分子质量约为25KDa,与预期分子量相符。免疫印迹在约25KDa有一明显特异印迹条带。转染细胞培养48h,SDS-PAGE分析,有相对分子质量约为27KDa目的重组蛋白表达,与预期的融合蛋白大小相符。ELISA检测到正常人、HIV阳性和HTLV-II阳性的参考血清均为阴性;HTLV-I阳性的参考血清为强阳性。结论 HTLV-I env基因5915nt-6545nt区,可以利用原核系统表达得到特异性HTLV-I重组抗原,重组抗原无显著性差异,有望成为检测试剂的抗原。
Objective To investigate the expression of recombinant env antigen I of human T-cell lymphotropic virus (HTLV). Methods The target gene of HTLV-I env was analyzed and cloned into the prokaryotic expression vector pQE80L. The recombinant plasmid was identified by PCR and restriction enzyme digestion. The purified recombinant env protein was induced by affinity chromatography and detected by Western-blot. Protein antigen activity, ELISA method to test the specificity of the recombinant env protein antigen. Results The positive recombinant pQE80L-env was obtained by PCR and enzyme digestion. SDS-PAGE showed that the relative molecular weight of the recombinant protein was about 25KDa, which was consistent with the expected molecular weight. Immunoblots have a distinctly specific blot at about 25 kDa. The transfected cells were cultured for 48h and analyzed by SDS-PAGE. The expressed recombinant protein with the molecular weight of about 27KDa was consistent with the size of the expected fusion protein. ELISA detected normal, HIV positive and HTLV-II positive reference serum were negative; HTLV-I positive reference serum is strongly positive. Conclusion The HTLV-I env gene 5915nt-6545nt region can express specific HTLV-I recombinant antigen by using prokaryotic system. The recombinant antigen has no significant difference and is expected to be the antigen of the detection reagent.