编码弓形虫表面抗原P30、P22复合基因真核表达载体的构建

目的　构建编码弓形虫RH株表面抗原P30、P22复合基因的真核表达重组质粒, 为进一步表达融合蛋白及研制核酸疫苗做准备。　方法　用弓形虫RH株腹腔接种小鼠,收集腹水,酚/氯仿法抽提弓形虫基因组 DNA;用 PCR技术从基因组DNA中扩增编码表面抗原 P30、P22 的基因片段,分别重组入 pMD18 T载体中。将 pMD18 T载体中的P30、P22基因片段分别酶切,定向克隆入 pUC18克隆载体中, pUC18 P30 P22 中的 P30 P22 片段经酶切、纯化后,亚克隆入 pcDNA3.1( )真核表达载体,用酶切、PCR及测序的方法对重组子进行鉴定。　结果　从弓形虫 RH株基因组DNA中扩增出特异的P30及P22片段;大小均与预测值相符;克隆 pUC18 P30 P22 重组质粒的酶切片段分别与 P30、P22基因大小一致;经亚克隆、筛选鉴定获得了 pcDNA3.1 P30 P22重组质粒,所测P30、P22基因序列与文献报道一致。结论　成功构建弓形虫 pUC18 P30 P22重组质粒和 pcDNA3.1 P30 P22 重组质粒,为研制弓形虫 DNA疫苗奠定了基础。

CONSTRUCTION OF EUKARYOTIC EXPRESSION RECOMBINANT PLASMID ENCODING P30 AND P22 COMPOUND GENE OF THE MAJOR SURFACE ANTIGEN OF TOXOPLASMA GONDII

Objective To construct eukaryotic expression recombinant plasmid encoding P30 and P22 compound gene of the major surface antigen of Toxoplasma gondii and to prepare for the research of fused protein expression and DNA vaccination. Methods T. gondii RH strain tachyzoites were injected into mice and ascites of infected mice were harvested. Genomic DNA of T. gondii was extracted with phenol/chloroform. The fragments encoding P30 gene and P22 gene were amplified respectively from genomic DNA of T. gondii RH strain by PCR, and the genes were inserted into pMD18 T vector respectively. Then the digested P30 gene and P22 gene were inserted into the cloning vector, pUC18. The inserted P30 P22 gene was recombined with pcDNA3.1( ) eukaryotic expression vector by digesting with restrictive enzymes and linking reactions. The positive clones were screened on LB plates containing ampicillin and identified by restrictive enzyme digestion, PCR amplification and sequencing. Results The specific fragments of P30 gene and P22 gene amplified from genomic DNA of T. gondii RH strain were of the same size as the predicted. The size of the digested fragments from the recombinant plasmid pUC18 P30 P22 was the same as the P30 gene and P22 gene. The recombinant plasmid pcDNA3.1 P30 P22 was constructed successfully by subcloning, screening and identifying. The sequences of the P30 gene and P22 gene were consistent with the published papers. Conclusion The recombinant plasmids pUC18 P30 P22 and pcDNA3.1 P30 P22 were constructed successfully.