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目的构建弓形虫致密颗粒蛋白GRA4的真核表达质粒,并研究其在体外与体内细胞中的表达情况。方法采用PCR方法及克隆技术,构建pVACGRA4重组质粒;利用脂质体介导转染法,将该重组质粒导入HEK293细胞,用RTPCR法及Westernblot法检测其表达情况;利用基因枪导入法将该重组质粒导入BALB/c小鼠体内细胞,测定免疫鼠T淋巴细胞亚群及IFNγ、IL4含量,并观察攻毒试验后小鼠存活情况,以评价其免疫保护力。结果从弓形虫RH株基因组DNA中扩增出GRA4基因片段,成功构建重组表达质粒pVACGRA4;转染GRA4的HEK293细胞,RTPCR检测可见一条约987bp的目的条带,表达产物经Westernblot鉴定,其分子量约为41kD,能被特异性免疫血清所识别;经pVACGRA4免疫后的小鼠,其CD+4T细胞百分率无明显变化(P>0.05),CD+8T细胞百分率明显增加(与pVAC对照组比较P<0.05,与空白对照组比较P<0.01),CD+4/CD+8比值也较空白对照组明显降低(P<0.01);pVACGRA4免疫鼠IFNγ的A值比对照组略高,但差异无显著性(P>0.05),IL4A值各组间无明显变化(P>0.05);pVACGRA4免疫鼠存活率显著高于两对照组,死亡小鼠的平均存活时间延长(与空白对照组比较P<0.05)。结论构建的真核表达质粒pVACGRA4能在HEK293细胞中和小鼠体内细胞中表达,为弓形虫疫苗的进一步研究打下基础。
Objective To construct the eukaryotic expression plasmid of Toxoplasma gondii GRA4 and study its expression in vitro and in vivo. Methods The recombinant plasmid pVACGRA4 was constructed by PCR and cloning techniques. The recombinant plasmid was transfected into HEK293 cells by lipofectamine 2000. The recombinant plasmid was transfected into HEK293 cells by RTPCR and Western blotting. The plasmids were introduced into BALB / c mice to determine the T lymphocyte subsets and IFNγ, IL4 levels in the immunized mice. The survival rate of the mice after the challenge test was observed to evaluate the immunopotency. Results The GRA4 gene fragment was amplified from the genomic DNA of RH strain Toxoplasma gondii and the recombinant plasmid pVACGRA4 was successfully constructed. The HEK293 cells transfected with GRA4 showed a 987bp band. The expressed product was identified by Western blot and its molecular weight was about (P <0.05). The percentage of CD + 8T cells was significantly increased (compared with pVAC control group, P < 0.05, compared with the blank control group, P <0.01), and the ratio of CD + 4 / CD + 8 was also significantly lower than that of the blank control group (P <0.01). The A value of IFNγ in pVACGRA4 immunized mice was slightly higher than that in the control group (P> 0.05). There was no significant difference in IL4A between the groups (P> 0.05). The survival rate of pVACGRA4 immunized mice was significantly higher than that of the two control groups, and the average survival time of the dead mice was prolonged (P <0.05 compared with the blank control group ). CONCLUSION: The constructed eukaryotic expression plasmid pVACGRA4 can be expressed in HEK293 cells and in vivo in mice, which lays a foundation for the further study of Toxoplasma gondii vaccine.