表达HIV-1gag蛋白重组乳酸乳球菌株的建立及其在小鼠体内定植的研究

Construction of the recombinant Lactococcus lactis expressing HIV-1 gag protein and its colonization in mice

目的构建表达HIV-1gag的重组乳酸乳球菌，并分析重组菌在小鼠体内的定植情况。方法将HIV-1gag克隆至原核表达质粒pMG36e，重组质粒pMG36e-gag电转乳酸乳球菌，SDS-PAGE和Western blot法检测其在乳酸乳球菌中的表达。以10^9个重组菌给小鼠口服，对照组口服PBS。口服后第1、3、5、7、9、11天分别取小鼠的胃、小肠和大肠，采用稀释滴种法测定小鼠胃、肠道内重组菌的数量。结果酶切和DNA测序表明gag已克隆人pMG36e。SDS—PAGE在预期位置检测到蛋白条带，Western blot分析证实其为gag蛋白。重组菌定植实验表明，第1、3天在胃肠均有一定量重组菌，且大肠中重组菌数量多于胃和小肠，随后胃肠中重组菌均迅速减少，仅在大肠中仍有一定量的重组菌，第11天时胃肠仅分离到极少量的重组菌。结论本研究获得胞内表达HIV-1gag前体蛋白Pr55的重组乳酸乳球菌，其可在胃肠中短期存活，可作为预防HIV-1感染的候选疫苗。

Objective To construct the recombinant Lactococcus lactis（ L. lactis） expressing HIV-I gag and research its colonization in mice. Methods HIV-1 gag was cloned into prokaryotic expression plasmid pMG36e, then the recombinant plasmid （pMG36e-gag） was electrotransformed into L. lactis. The expression of gag in L. lactis was analyzed by SDS-PAGE and Western blotting, respectively. The experimental mice were administrated orally with the 1 x 109 L. lactis, and the control mice with PBS instead. At 1,3, 5, 7, 9 and 11 d after administration, the mice were sacrificed, and then the stomach, small intestine and large intestine were taken from each mouse. The L. lactis number of colonization in these tissues were assayed by means of diluting and dripping. Results Restriction endonuclease analysis and DNA sequencing confirmed that gag had been cloned into the plasmid pMG36e. SDS-PAGE and Western blotting identified the gag antigen in L. lactis as we expected. At 1 and 3 d, there were some colonized recombinant L. lactis in the stomach and intestine, and the number of L. lactis colonized in the large intestine was higher than that in the stomach and small intestine. Thereafter, the number of L. lactis was rapidly reduced in the stomach and intestine, and finally only in large intestine a few still existed. At tl d, only a few recombinant L. lactis was isolated from the stomach and intestine. Conclusion The recombinant L. lactis expressing HIV- 1 gag precursor protein （Pr55） was constructed, and it can survive for a short time in the stomach and intestine, so it could act as a candidate vaccine to prevent HIV-1 infection.