单纯疱疹病毒I和Ⅱ型共同抗原gD在大肠埃希菌中的重组表达

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-c^2/gD表达质粒，Western blot证实，HSV－I gD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。

Cloning and expression of HSV-I, II type-common antigen gD in Escherichia coli]

BACKGROUND: To clone the type common antigen gD of human herpes simplex virus I, II (HSV-I, II), the authors constructed recombinant expression vector Pmal-c2/gD and induced to express the fusion protein MBP-gD. METHODS: The authors extracted HSV DNA,amplified gD gene by PCR assay and directly cloned it into prokaryotic expression vector pMAL-c2, then transformed it into E.coli DH5alpha. After proved to be correct by PCR, double enzyme digestion and sequencing, the fusion protein is induced to express by IPTG and detected by both Western blot and ELISA. RESULTS: The constructed expression vector pMAL-c2/gD can be expressed with high efficiency. The product expressed was about 35.5% of the total bacterium proteins by SDS?PAGE analysis and was found nearly 39% as soluble protein,61% as inclusion in cytoplasm. CONCLUSIONS: The authors constructed recombinant expression vector pMAL-c2/gD, the Western blotting result showed that the recombinant protein could be identified with gD specific monoclonal antibody DL6. Therefore the protein was of natural antigenic structure of gD.