刚地弓形虫RH株GRA6分子基因克隆与序列分析

CLONE AND SEQUENCE OF THE DENSE GRANULE ANTIGEN (GRA6) OF THE TOXOPLASMA GONDII RH STRAIN

目的 克隆刚地弓形虫 RH株致密颗粒抗原 (Dense granule antigen,GRA6 )分子基因 ,为研究使用重组的GRA6分子作为抗原进行弓形虫病诊断奠定基础。 方法 根据己知的 GRA6序列合成一对引物 ,抽提弓形虫速殖子m RNA,以速殖子 m RNA为模板 ,通过反转录 PCR(RT- PCR)扩增出 GRA6的 c DNA条带。目的基因 DNA条带被克隆到质粒 p UC19中形成重组质粒。对重组质粒 DNA进行纯化 ,并对目的基因片段进行核苷酸序列分析。 结果 通过RT- PCR从 m RNA中扩增出一条分子量约 70 0 bp的 DNA条带。核苷酸序列分析表明 ,GRA6分子基因的开放的阅读框全长为 6 93bp,编码 2 30个氨基酸分子 ,与已报道的 RH株 GRA6分子具有 10 0 %的同源性。 结论 本研究获得了刚地弓形虫 RH株的 GRA6分子基因 。

Objective To clone the gene of dense granule antigen (GRA6) of Toxoplasma gondii RH strain and investigate the value of recombinant GRA6 as a diagnostic antigen of toxoplasmosis. Methods A pair of primers was synthesized according to the DNA sequence of GRA6 of T. gondii RH strain, and the mRNA of tachyzoites of RH strain was prepared. This gene fragment was cloned into the plasmid pUC19 to form the recombinant. The recombinant plasmid was purified and the target DNA fragment was sequenced. Results A single?specific DNA fragment (approximately 700 bp) was amplified from the genomic DNA of tachyzoites of T. gondii successfully. The result of DNA sequencing showed that the Open Read Frame of GRA6 is composed of 693 base pairs and encodes 230 amino acid, which has 100% homology to the reported sequence of GRA6 of T. gondii RH strain. Conclusion The gene of GRA6 of T. gondii RH strain has been cloned successfully, which make a good basis for using GRA6 as diagnostic antigen to diagnosis toxoplasmosis.