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目的 克隆人单纯疱疹病毒Ⅰ、Ⅱ (HSV Ⅰ、Ⅱ )型共同性抗原gD基因 ,构建重组表达载体pMAL c2 gD ,诱导融合蛋白MBP gD的表达。方法 提取病毒DNA ,PCR扩增出gD基因 ,克隆于原核表达载体pMAL c2并转化大肠埃希菌DH5α。PCR、双酶切及测序证实插入的gD基因序列正确后 ,IPTG诱导表达融合蛋白MBP gD ,并进行免疫学鉴定。结果 构建的重组表达质粒pMAL c2 gD在大肠埃希菌中能高效表达。经SDS PAGE分析 ,表达产物约占菌体总蛋白 35 5 % ,其中 39%以可溶蛋白形式存在于胞质中 ,61 %以包涵体形式存在。结论 构建了pMAL c2 gD表达质粒 ,Westernblot证实 ,HSV ⅠgD单克隆抗体DL6可特异识别表达的gD蛋白 ,该蛋白具有天然gD的抗原性
Objective To clone the gD gene of human herpes simplex virus Ⅰ, Ⅱ (HSV Ⅰ, Ⅱ) common antigen and construct the recombinant expression vector pMAL c2 gD to induce the expression of fusion protein MBP gD. Methods The virus DNA was extracted and the gD gene was amplified by PCR. The recombinant plasmid was cloned into prokaryotic expression vector pMAL c2 and transformed into Escherichia coli DH5α. After PCR, double digestion and sequencing confirmed that the sequence of inserted gD gene was correct, IPTG induced expression of the fusion protein MBP gD, and immunological identification. Results The constructed recombinant plasmid pMAL c2 gD was highly expressed in Escherichia coli. According to SDS PAGE analysis, the expression product accounted for about 35.5% of total bacterial proteins, of which 39% existed as soluble proteins in cytoplasm and 61% as inclusion bodies. Conclusion The pMAL c2 gD expression plasmid was constructed. Western blot confirmed that the monoclonal antibody DLV of HSV Ⅰ gD specifically recognizes the expressed gD protein, which has the antigenicity of natural gD