对嗜T淋巴细胞病毒Ⅰ型重组env的研究

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。

Study on the expression of recombinant antigen env in human T-cell lymphotropic virus type into prokaryotic vector I

Objective To investigate the expression of recombinant antigen env in human T-cell lymphotropic virus type I.Methods The target gene of HTLV-I env were analyzed and selected,cloned into prokaryotic vector pQE80L and identified it through PCR and restriction methods.Then the expressed recombinant env protein antigen was induced and purified by affinity chromatography.Western-blot method was adapted to test the activity of the recombinant env antigen expressed by prokaryotic,and ELISA method was adapted to test its specificity.Results The positive recombinants pQE80L-env was selected through PCR amplification and restriction.SDS-PAGE suggested the relative molecular mass of the recombinant protein was approximately 25KDa,which was in line with the expected molecular weight,and Western-blot showed an obvious specific band at 25KDa.The transfected cells were cultured for 48h,then the corresponding SDS-PAGE indicated the relative molecular mass of the recombinant protein was approximately 27KDa,which coincided with the expected molecular weight,and Western-blot revealed an obvious specific band at 27KDa.The reference sera of normal,HIV-positive and HTLV-Ⅱ-positive people were all detected negative,while HTLV-Ⅰ-positive people was strongly positive.Conclusion 5915-6545nt area of HTLV-I env gene can be cloned into prokaryotic and eukaryotic vector to express the specific recombinant antigen HTLV-I.There are no significant differences between the two antigens,and it holds the potential to be used as test kits.