狂犬病毒糖蛋白基因-腺病毒E3区缺失转移表达载体的构建及表达

目的构建狂犬病毒糖蛋白基因-腺病毒E3缺失转移表达载体,并在MDCK细胞系统中高效表达。方法将含狂犬病毒SRV9株糖蛋白(Rgp)cDNA的pGT载体,经双酶切后回收Rgp基因片段,与经双酶切的真核表达载体pIRES1neo连接,得到pIRES1Rgp,再经双酶切后回收2623bp片段,与E3缺失载体pBE3L连接,得到含狂犬病毒糖蛋白基因-腺病毒E3缺失转移表达载体pBE3LCRgp,再转染MDCK细胞。结果pBE3LCRgp转染的MDCK细胞,经间接ELISA和间接免疫荧光法在其培养上清中均能检出Rgp的高效表达。结论已成功构建狂犬病毒糖蛋白基因-腺病毒E3缺失转移表达载体,并在MDCK细胞中高效表达。

Construction of Recombinant Canine Adenovirus Type-2 with E3 Region Deleted as A Transfer Expression Vector of Rabies Virus Glycoprotein

Objective To construct the recombinant canine adenovirus type-2 with E3 region deleted as a transfer expression vector and highly express rabies virus glycoprotein in MD CK cell system. Methods Digest the pGT vector containing the cDNA encoding the glycoprotein(Rgp) of SRV9 strain of rabies virus with XbaⅠ and SalⅠ,recover Rgp gene fragment and inser t into eukaryotic expression vector pIRES1neo.Digest the recombinant plasmid pIR ES1Rgp with SalⅠ and NruⅠ to obtain a 2 623 bp fragment.Ligate the fragment to vector pBE3L with E3 region deleted to generate a recombinant canine adenovirus type-2 with E3 region deleted as a transfer expression vector pBE3LCRgp of rab ies virus glycoprotein for the transfection of MDCK cells. Results The high expression of Rgp was detected in culture supernatant of MDCK cells tra nsfected with pBE3LCRgp by ELISA and indirect immunofluorescence method. Conclusion A canine adenovirus type-2 E3 region transfer vector expressing rabies virus gl ycoprotein was successfully constructed,and rabies virus glycoprotein was highly expressed.