摘要
目的构建HIV-1HXB2株ta(tB41-101N)融合基因原核表达质粒,表达并纯化Ta(tB41-101N)融合蛋白,为进一步研究其免疫原性奠定基础。方法在HIV-1HXB2株天然Tat蛋白N-端添加Tat41-101位氨基酸(aa),采用PCR法从HIV-1HXB2株tat基因中扩增分别编码Tat41-101aa和Tat1-101aa的tat41-101和tat1-101两个基因片段,重叠延伸PCR法扩增其融合基因ta(tB41-101N),并构建其原核表达质粒pET32a-ta(tB41-101N),经双酶切及测序验证后,转化大肠杆菌BL21(DE3),IPTG诱导表达。融合蛋白经Ni2+-NTA柱亲和层析纯化后,进行SDS-PAGE、Westernblot及ELISA鉴定。结果重叠延伸PCR扩增出约500bp的ta(tB41-101N)融合基因,酶切及测序结果表明重组表达质粒pET32a-ta(tB41-101N)构建正确。SDS-PAGE分析显示,表达的Ta(tB41-101N)融合蛋白相对分子质量约为36000,约占菌体总蛋白的7%,纯化后纯度约为60%;Westernblot和ELISA分析显示,Ta(tB41-101N)融合蛋白与小鼠抗Tat单抗及抗-HIV阳性血清(抗Tat抗体阳性)均呈特异性阳性反应。结论已成功构建了HIV-1HXB2株ta(tB41-101N)融合基因原核表达质粒,并表达了其融合蛋白,该蛋白较好地保留了天然Tat蛋白的免疫反应性,为Tat新型疫苗的研究奠定了基础。
Objective To construct the prokaryotic expression vector for tat（B41-101N）fusion gene of HIV-1 HXB2 strain, express and purify Tat （B41-101N）fusion protein and lay a foundation of study on its immunogenicity. Methods Tat41-101 amino acids（aa）were added to the N-terminus of Tat protein of native HIV-1 HXB2 strain. The tat41-101 and tat1-101 gene fragments, encoding Tat41-101aa and Tat1-101aa respectively, were amplified by PCR and used to generate fusion gene tat（B41-101N）by splicing by overlap extension PCR （SOE PCR）. Prokaryotic expression vector pET32a-tat （B41-101N） was constructed and identified by restriction analysis and sequencing, then transformed to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed filsion protein Tat （B41-1OIN） was purified by Ni2＋-NTA column affinity chromatography and identified by SDS-PAGE, Western blot and EI,ISA. Results The fusion gene tat（B41-101N） at a length of about 500 bp was amplified by SOE PCR. Recombinant ptasmid pET32a-tat （B41-101N） was proved to be constructed correctly by both restriction analysis and sequencing. SDS-PAGE showed that Tat （B41-101N ） fusion protein, with a relative molecular mass of about 36 000, was expressed, which contained about 7% of total somatic protein and reached a purity of about 60% ＇after purification. Tat（ B41-101N ） fusion protein showed specific reaction with mouse McAb against Tat and anti-HIV positive sera, as proved by Western blot and ELISA. Conclusion The prokaryotic expression vector for tat （B41-101N） fusion gene of HIV-I HXB2 strain was successfully constructed, and the fusion protein with immunoreactivity of native Tat protein was expressed, which laid a foundation of further study on novel HIV Tat vaccine.
出处
《中国生物制品学杂志》
CAS
CSCD
2009年第11期1049-1053,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金资助项目(30872246
30872405)
国家"863"项目(2006AA02A238)
上海市基础研究重点项目(08JC1405200)
