刚地弓形虫亲环蛋白基因TgCyP真核表达质粒的构建及其在Hela细胞内的表达

亲环蛋白（cyclophilin，CyP）是一类广泛存在于原核和真核生物体内的胞溶性蛋白，是刚地弓形虫（Toxoplasmngondii）速殖子的主要成分，能够诱导产生IL-12和IFN-γ，在控制弓形虫急性感染过程中起重要作用。本研究根据GenBank发表的TgCyP基因序列，设计并合成一对包含BamHI和EcoRI酶切位点的引物，以cDNA为模板，应用PCR技术扩增TgCyP基因。PCR产物连接到pMD18-T克隆载体。用限制性内切酶BamHI和EcoRI双酶切克隆载体，将TgCyP目的基因克隆到载体pVAXI，构建真核表达质粒pVAXl-TgCyP。将真核表达质粒转染Hela细胞，利用问接免疫荧光检测其在Hela细胞内的表达情况。结果表明扩增的TgCyP基因与GenBank上相应基因序列（U04633．1）的一致性达100％，构建的真核表达质粒pVAX1-TgCyP能在转染的Hela细胞中表达，其表达产物与刚地弓形虫阳性血清具有免疫反应性。本研究表明Tg-CyP有望作为弓形虫疫苗的候选抗原，将为进一步研究该质粒的动物免疫试验奠定基础。

Construction of the Eukaryotic Expression Plasmid of TgCyP Gene from Toxoplasma gondii and Its Expression in Hela Cells

Cyclophilins （CyPs） are ubiquitous cytosolic proteins and have been described in prokaryote as well as eukaryote. TgCyP is a critical tachyzoite constituent of T. gondii. It can induce the production of IL-12 and IFN-γ and may be play an important part in the process of controlling acute phase oftoxoplasmosis. In this study, according to TgCyP gene sequence published in GenBank, a pair of specific primers were designed and synthesized included a BamH I and EcoR I restriction enzyme site. The eDNAs were used as templates for amplification of the sequences of recombinant TgCyP by PCR. Then, TgCyP gene fragments were transformed into pMD18-T vector. After cloned, the vector was digested with BamH I and EcoR I and then ligated into plasmid pVAX1, generated the eukaryotic expression plasmid pVAX1-TgCyP. Then, the eukaryotic expression plasmid pVAX1-TgCyP was transfected into Hela cells. Recombinant protein expression from this plasmid in Hela cells were confirmed by indirect immunofluorescence staining. The results showed that DNA sequence identity was 100% between amplified TgCyP and amino acid sequences of TgCyp which were stored in the GenBank database under accession number U04633.1. The indirect immunofluorescence test showed that the eukaryotic expression plasmid was expressed in Hela cells and recognized by T. gondii positive serum, which might be used as a candidate antigen of T. gondii vaccine. These available data would lay the foundation for further studying on DNA vaccine against T. gondii.