弓形虫新基因表位疫苗质粒的构建及鉴定

The construction and characterization of multiple-epitopes vaccine of two new genes of Toxoplasma gondii

目的构建两个弓形虫新基因2B9G1、7C3-C3的表位疫苗质粒,为阐明表位疫苗的保护性奠定基础。方法采用生物信息学方法对新基因2B9G1、7C3-C3进行表位预测,PCR扩增基因中编码3个表位的片段W2b、W2a和W4a,连接入pcDNA3,转入DH5α,挑阳性菌落进行PCR、酶切和测序鉴定;同法将W2a、W4a分别克隆入pcDNA3-W2b载体中W2b片段下游,再将W4a克隆入pcDNA3-W2b2a载体中W2a片段下游。结果经PCR、酶切鉴定及序列测定,W2b、W2a和W4a 3个片段分别插入pcDNA3预期位置;W2a和W4a片段分别插入pcDNA3-W2b预期位置;W4a片段插入pcDNA3-W2b2a预期位置。结论成功构建单表位、双表位、多表位疫苗质粒pcDNA3-W2b、pcDNA3-W2a、pcDNA3-W4a、pcDNA3-W2b2a、pcDNA3-W2b4a、pcDNA3-W2a2b4a。

Objective To construct a series of vaccine vectors carrying epitopes from two novel genes 2B9G1 or 7C3-C3 of Toxoplasma gondii and lay the foundation for further research on protection induced by them. Methods The epitopes encoded by sequences within two novel genes were predicted by bioinformatics. Three epitope encoding fragments W2b, W2a and W4a were selected, and amplified from either 2B9G1 or 7C3-C3 gene by PCR, respectively. The amplified products were purified with DNA purification kit and then digested with restriction endonucleases. Digested products were then ligated into corresponding polyclone sites of plasmid with T4 DNA ligase respectively. The recombinants were transformed into E. coli DH5α-cells by heat-shock method. Ampicillin-resistant transformants were then selected and identified by PCR, restriction endonucleases and DNA sequencing analysis. Identically, W2a and W4a were cloned into the downstream of recombinant pcDNA3-W2b, respectively, and W4a into the downstream of recombinant pcDNA-W2b2a. Results With the confirmation by the PCR, restriction endonucleases and DNA sequencing analysis, the epitopes of W2b, W2a and W4a were inserted into the expecting site of pcDNA3 ; W2a and W4a were also inserted into pcDNA3-W2b and W4a into pcDNA3-W2b2a, respectively. Conclusion By these procedures, the single-epitope vaccine plasmid pcDNA3- W2b, pcDNA3-W2a, pcDNA3-W4a and double-epitope vaccine pcDNA3-W2b2a, pcDNA3-W2b4a, and multiple-epitope vaccine pcDNA3-W2a2b4a were successfully constructed.