表达VP3重组减毒沙门氏菌的构建和融合蛋白纯化

Construction of VP3-expressing recombinant attenuated Salmonella typhimurium vaccine strain and the purification of VP3 fusion protein

寻求有效的肿瘤基因疗法,构建鸡贫血病毒VP3的减毒鼠伤寒沙门氏菌疫苗,并获得较纯的表达VP3基因的融合蛋白,初步研究其免疫原性。采用PCR技术扩增VP3基因,并将其与原核载体pET32α(+)重组。将重组后质粒转染E.coliBL21,得到表达VP3的融合蛋白,并将此蛋白通过50%的Ni+-NTA亲和树脂纯化。同时将重组质粒转染减毒沙门氏菌SL7207。经双酶切和PCR鉴定,成功构建了表达VP3的减毒沙门氏菌苗。融合蛋白通过50%的Ni+-NTA亲和树脂纯化,得到纯度在90%以上的纯化蛋白。成功构建了表达VP3的减毒沙门氏菌苗,并且获得纯化的表达VP3的融合蛋白,为进一步研究VP3的免疫保护作用及对抗肿瘤疫苗的研制打下基础。

To construct the recombinant attenuated live Salmonella typhimurium vaccine strain expressing VP3 and purify the fusion protein of Trx-His-VP3 and study its immunactivity. Amplified VP3 gene was inserted into pET32α（＋） plasmid by T4 DNA ligase,and the recombinant plasmid was transformed into E. coli BL21 cells. The protein expression was induced by IPTG ,and fusion proteins were purified by 50% Ni＋ -NTA on a 5 mL column, then its immunoreactivity was identified. Meanwhile the identified recombinant plasmid was transformed into an attenuated Salmonella typhimurium vaccine strain SL7207, and positive clones were screened by PCR and restriction enzyme digestion. The expression of fusion protein Trx-His-VP3 was analyzed by SDS-PAGE and Western blot. A fragment of 366bp VP3 DNA was amplified by PCR. The recombinant plasmid was confirmed by sequencing. The recombinant attenuated Salmonella typhimurium vaccine strain SL7207 was constructed successfully. VP3 was expressed in the recombinant strains, and its immunogenicity was confirmed by Western blot. The fusion proteinTrx-His-VP3 of 34kD was expressed and purified. A recombinant attenuated live Salmonella typhimurium vaccine strain expressing VP3 was constructed and identified, the fusion protein Trx-His-VP3 with high purity was obtained, and its immunoreactivity was studied.