刚地弓形虫棒状体蛋白ROP16对小鼠脑神经细胞凋亡的影响

Toxoplasma gondii ROP16 causes apoptosis of mouse brain nerve cells

目的构建带有Flag标签的刚地弓形虫(Toxoplasma gondii)棒状体蛋白16(ROP16)的重组慢病毒表达质粒,并检测ROP16蛋白在神经细胞中的表达及其对小鼠脑神经细胞凋亡的影响。方法PCR扩增带Flag标签的ROP16基因,克隆至慢病毒载体,构建重组质粒p CDH-cop GFP-ROP16-Flag,转化至大肠埃希菌(Escherichia coli)DH5α感受态细胞,采用PCR、双酶切及测序验证。将重组质粒转染至人胚胎肾上皮细胞(293T细胞)中进行慢病毒包装,以p CDH-CMV-cop GFP空质粒对照组,荧光显微镜下观察绿色荧光表达情况,并计算慢病毒滴度。将包装后的慢病毒感染人神经母细胞瘤细胞(SH-SY5Y),采用蛋白质印迹(Western blotting)检测ROP16蛋白的表达情况。应用立体定位注射技术将慢病毒浓缩液注射至小鼠前额叶皮层M1区,2周后取小鼠脑组织,制备冰冻切片,TUNEL染色后,荧光显微镜下观察小鼠脑神经细胞凋亡情况。结果重组慢病毒表达质粒p CDH-cop GFP-ROP16-Flag经PCR和双酶切后均获得大小为2 200 bp的条带,与预期值相符;测序结果与刚地弓形虫RH株ROP16基因(Gen Bank登录号为GQ249093.1)序列比对完全一致。包装后的慢病毒在荧光显微镜下可见绿色荧光,滴度约为2E+8 TU/ml。Western blotting分析结果显示,在相对分子质量(Mr)120 000处有特异条带,重组慢病毒表达质粒能够在SH-SY5Y细胞内表达ROP16蛋白。感染慢病毒的小鼠脑组织冰冻切片经TUNEL染色后,荧光显微镜下可见实验组凋亡的细胞明显多于空质粒对照组。结论构建的慢病毒表达质粒p CDH-cop GFP-ROP16-Flag可在神经细胞内表达ROP16蛋白,并可导致小鼠脑神经细胞凋亡。

Objective To construct a recombinant lentivirus expression vector containing Flag-tagged sequence of Toxoplasma gondii ROP16, and test its expression in nerve cells and its effect on apoptosis of brain nerve cells in mice. Methods The Flag-tagged ROP16 gene was amplified by PCR and cloned into lentiviral vector to construct the recombinant plasmid pCDH-CMV-copGFP-ROP16-Flag, which was then transformed into E.coli DH5a competent cells. The recombinant plasmids were verified with PCR, double enzymetic digestion and sequencing. Then they were packaged into virus to infect 293T cells. The green fluorescence was observed under a fluorescence microscope and the virus titer was calculated. The packaged lentivirus was then used to infect SH-SY5Y cells, accompanied by a pCDH-CMV-eopGFP empty plasmid group. Expression of ROP16 was examined by Western blotting. The concentrated lentivirus was injected into the mouse prefrontal cortex M1 region by stereotactic injection. Two weeks later, the mice were sacrificed and frozen slices of the brain were prepared. Apoptosis of brain nerve cells was observed under a fluorescence microscope after TUNEL staining. Results As expected, PCR and double enzymetic digestion both resulted in a band of--2 200 bp from the recombinant plasmid pCDH-copGFP-ROP16-Flag. Sequencing result was consistent with the ROP16 gene of Toxoplsma gondii RH strain （GenBank Accession No. GQ249093.1）. Green fluorescence was seen under the fluorescence microscope with a titer of about 2E ＋ 8 TU/ml. Western blotting analysis showed a specific band at a relative molecular weight of 120 000, suggesting that the recombinant plasmid could express ROP16 protein in SH-SY5Y cells. TUNEL staining revealed significantly moreapoptotic cells in the ROP16 group than other groups. Conclusion The lentiviral vector pCDH-copGFP-ROP16-Flag expresses ROP16 protein in nerve cells and induces apoptosis of mouse brain nerve cells.