摘要
目的 构建弓形虫RH株致密颗粒抗原GRA8的原核和真核重组表达质粒。方法 参照GRA8序列分别设计引物 ,采用PCR从弓形虫RH株基因组DNA中分别扩增出编码GRA8的基因片段 ,克隆至 pMD18-T载体 ;菌落PCR鉴定阳性克隆并测序分析 ;各组阳性克隆的质粒分别亚克隆至原核表达质粒pGEX - 4T - 2和真核表达载体 pVAX1,分别转化大肠杆菌BL2 1和JM10 9,PCR和酶切鉴定转化菌落的插入序列 ;将构建的原核表达菌株经IPTG诱导 ,SDS -PAGE和免疫印迹分析融合蛋白的表达 ;将构建的真核重组表达质粒免疫小鼠 ,观察其诱导的抗体应答。结果 PCR扩增出GRA8基因的特异片段 ,各组阳性克隆的序列正确 ,并分别被亚克隆到原核表达质粒 pGEX - 4T - 2和真核表达载体pVAX1上 ,构建了弓形虫致密颗粒抗原GRA8的原核和真核重组表达质粒 ;原核表达质粒在大肠杆菌中表达了GRA8的融合蛋白 ;真核重组表达质粒诱导小鼠产生了抗弓形虫抗原的抗体。结论 以pGEX - 4T - 2和 pVAX1为载体 ,分别成功构建了GRA8的原核和真核重组表达质粒。
To construct the prokaryotic and eukaryotic expression plasmids containing the dense granule antigen GRA8 gene of Toxoplasma gondii RH strain,the gene fragment encoding GRA8 was amplified by PCR with special primers from genomic DNA of Toxoplasma gondii and was cloned into pMD18-T vector.The positive clones were identified by colony PCR,and undergone sequencing with ABI PRISM TM 377LX DNA sequencer.The right gene fragments encoding GRA8 in positive clones for prokaryotic and eukaryotic expression plasmid were digested with restrictive endonuclease,and were subcloned into pGEX-4T-2 and pVAX1 respectively.Then,the recombinant prokaryotic and eukaryotic plasmids(pGEX-GRA8 and VAX1-GRA8) were transformed into E.coli BL21 and JM109 respectively.The positive recombinant clones were characterized with PCR and digested with restrictive endonucleases and the pGEX-GRA8 in BL 21 was induced with IPTG to express target protein,which could be characterized by SDS-PAGE and Western blotting analysis.BALB/c mice were immunized with pVAX1=GRA8 so as to evaluate the humoral immune responses.The experimental results showed that the specific fragment of GRA8 had been amplified by PCR with correct sequences in the positive clones and could be subcloned into prokaryotic expression vector pGEX-4T-2 and eukaryotic expression vector pVAX1 respectively.The inserts of GRA8 in two groups of positive clones were coincident with the original sequences of GRA8 gene from GenBank,and the pGEX-GRA8 and pVAX1-GRA8 were constructed through subcloning the right inserts of GRA8 into pGEX-4T-2 and pVAX1 respectively.Recombant fusion protein was expressed in colonies containing pGEX-gra8 when induced with IPTG,and it reacted with the goat polyclonal antibodies to Schistosoma japonicum 26kDa GST.Mice immunized with pVAX1-GRA8 developed antibodies to Toxoplasma antigen with a titre of 1∶100.It concludes that the prokaryotic and eukaryotic expression vectors for GRA8 gene of Toxoplasma gondii have been successfully constructed.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2004年第3期203-206,210,共5页
Chinese Journal of Zoonoses
