刚地弓形虫RH株ROP18-ROP12复合基因真核表达载体的构建

Construction of Eukaryotic Expression Vector Containing ROP18-ROP12 of Toxoplasma gondii RH Strain

目的构建刚地弓形虫(Toxoplasma gondii)棒状体蛋白18(ROP18)与ROP12的复合基因真核表达重组质粒,并检验其在真核细胞中的表达情况。方法重组质粒p VAX1-ROP18和p VAX1-ROP12分别经Bam HⅠ和XbaⅠ双酶切,将ROP12基因克隆至p VAX1-ROP18重组质粒。经菌落PCR、酶切及测序鉴定正确的重组表达质粒p VAX1-ROP18-ROP12转染至He La细胞。同时设空质粒组、p VAX1-ROP18转染组和p VAX1-ROP12转染组。提取各组He La细胞的总RNA并逆转录为c DNA,分别进行管家基因β-肌动蛋白和ROP18-ROP12复合基因的RT-PCR鉴定;同时,采用间接荧光免疫法和蛋白质印迹(Western blotting)法检测重组质粒p VAX1-ROP18-ROP12瞬时转染He La细胞后蛋白的表达情况。结果重组质粒p VAX1-ROP18-ROP12的菌落PCR电泳显示在约2 373 bp处出现特异性扩增片段,与预期大小相符。提取重组质粒经HindⅢ、Bam HⅠ和XbaⅠ单酶切、双酶切及三酶切鉴定均正确。测序结果显示,p VAX1-ROP18-ROP12重组质粒与已发表的弓形虫RH株ROP18基因(登录号为AM075204.1)序列一致性为100%,与弓形虫RH株ROP12基因(登录号为DQ096559.1)序列一致性为99%。脂质体转染后各组β-肌动蛋白的RT-PCR扩增产物均为613 bp,与预期大小相符。p VAX1-ROP18-ROP12转染组的ROP18-ROP12复合基因扩增产物大小为2 373 bp,而其他组未见条带。间接荧光法检测显示,在重组质粒转染的He La细胞胞浆中观察到黄绿色荧光,而对照组无黄绿色荧光。Western blotting法检测显示,融合蛋白ROP18-ROP12相对分子质量(Mr)约为85 000。结论构建了重组质粒p VAX1-ROP18-ROP12,该质粒能在真核细胞中表达。

Objective To construct a recombinant eukaryotic expression plasmid containing ROP18-ROP12 （encoding rhoptry protein 18 and 12） complex gene of Toxoplasma gondii, and examine its expression in eukaryotic cells. Methods Recombinant plasmids pVAX1-ROP18 and pVAX1-ROP12 were digested by restriction enzymes BamH I and Xba I. ROP12 gene was cloned into pVAX1-ROP18 to construct the eukaryotic expression plasmid pVAX1-ROP18- ROP12. After colony PCR, enzyme digestion and sequencing, the correct recombinant plasmid pVAX1-ROP18-ROP12 was transfected into HeLa cells. Along with it were groups of empty plasmid, pVAX1-ROP18 and pVAX1-ROP12. Total RNA was extracted from HeLa cells and reverse-transcribed to cDNA. RT-PCR was performed to evaluate mRNA expression of the housekeeping gene [3-actin and ROP18-ROP12 complex gene. Immunofluorescence assay and Western blotting were performed to determine the protein levels of ROP18-ROP12 fusion protein. Results Colony PCR in recombinant plasmid pVAX1-ROP18-ROP12 showed a specific band at about 2 373 bp, consistent with expectation. The extracted recombinant plasmids were confirmed by Hind Ⅲ, BamH I and Xba I digestion. Sequencing results showed that the sequence of pVAX1-ROP18-ROP12 was 100% identical to that of T. gondii RH strain ROP18 gene （Accession No. AM075204.1） and 99% identical to that of T. gondii RH strain ROP12 gene （Accession No. DQ096559.1）. Further,RT-PCR showed amplification products at 613 bp for β-actin in all the groups, while only the pVAX1-ROP18-ROP12 transfection group showed amplification products for the ROP18-ROP12 complex at 2 373 bp. In addition, the indirect immunofluorescence assay showed yellow-green fluorescence in HeLa cells transfected with pVAX1-ROP18-ROP12, but not in control cells. Western blotting showed that the ROP18-ROP12 fusion protein was expressed in HeLa cells transfected with recombinant plasmid pVAX1-ROP18-ROP12. Conclusions The recombinant eukaryotic plasmid pVAX1- ROP18-ROP12 is constructed and can be expressed in eukaryotic system.