密码子优化的HIV-1型gp120基因重组腺病毒载体疫苗的构建与鉴定

CONSTRUCTION AND CHARACTERIZATION OF A CODON OPTIMIZED HIV-1 GP120 GENE RECOMBINANT ADENOVIRUS VECTOR

目的构建密码子优化型gp120基因重组腺病毒载体(rAden-mgp120),为研发新型HIV预防性疫苗奠定基础。方法首先利用PCR的方法扩增mgp120基因,将目的基因克隆入载体pENTR/D-TOPO以获得重组穿梭载体克隆pENTR/D-TOPO-mgp120。经过穿梭载体克隆与表达载体(pAd-CMV/V5-DEST)间的重组反应获得表达克隆质粒pAden-mgp120,表达克隆线性化后转染HEK293A包装细胞,得到复制缺陷型重组腺病毒rAden-mgp120。结果经PCR和测序鉴定,重组载体rAden-mgp120构建正确,转染HEK293A细胞并扩增后获得的病毒滴度为6.8x1010pfu/ml,该重组腺病毒载体能正确表达mgp120蛋白。结论成功构建了重组腺病毒rAdenmgp120为HIV-1预防奠定了基础。

Objective To generate recombinant adenoviral vector containing mgp120 gene for developing a safe, effective vaccine. Methods The mgp120 gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA （CACC） sequence was added to th＇e 5＇ end. Adenoviral expression vector containing mgp120 gene was constructed by homologous recombination. The linearized DNA plasmid of the recombi- nant adenoviral vector was transfected human embryo kidney （HEK） 293A cells to package and amplify re- combinant adenovirus. The recombinant adenovirus titer was characterized by using the End-dilution as- say. The expression of the mgp120 protein in 293A cells was detected by Western blot. Results The construction of mgp120 gene recombinant pENTR/D-TOPO transfer vector was successful. The recombinant adenoviral vector, Aden-mgpl20, was generated successfully, too. The titer of Aden-mgp120 was characterized as 6.8）〈 101~pfu/mL. The mgp120 protein was expressed by HEK 293A cells correctly. Conclusion The mgp120 gene recombinant replication-defective adenovirus expression vector was constructed successfully and this study has provided an experimental basis for further research on HIV-1 vaccine.