According to the published gene sequence of the major surface antigen (P30) of Toxoplasma gondii, a pair of primers were designed and synthesized. Using polymerase chain reaction (PCR), the coding sequences of P30 gen...According to the published gene sequence of the major surface antigen (P30) of Toxoplasma gondii, a pair of primers were designed and synthesized. Using polymerase chain reaction (PCR), the coding sequences of P30 gene were amplified from a Chinese strain of T. gondii, The amplified gene fragment and plasmid pB220 were digested with EcoRI and BamHI and then ligated. The inserted gene fragment was sequenced by the chain termination method, the reading reveals that nucleotide sequence determined was the same as the P30 sequecne of RH strain pubilished by Burg (1988), except that one base was changed. The recombinant plasmid containing P30 gene was transformed to E. coli DH5α.After temperature inducing culture, the total cellular proteins were analysed by SDS-PAGE and Western blot. The results show that the p30 gene cloned into the plasmid could express in E. coli, and the expression product had immunogenicity.展开更多
目的进行弓形虫棒状体蛋白2(ROP2)和膜表面蛋白1(P30)融合基因的克隆与表达,为弓形虫ROP2-P30基因工程复合抗原的制备做准备。方法半套式PCR扩增编码弓形虫P30的基因片段,克隆至已构建成功的重组质粒pUC119/ROP2中,经PCR和酶切鉴定正确...目的进行弓形虫棒状体蛋白2(ROP2)和膜表面蛋白1(P30)融合基因的克隆与表达,为弓形虫ROP2-P30基因工程复合抗原的制备做准备。方法半套式PCR扩增编码弓形虫P30的基因片段,克隆至已构建成功的重组质粒pUC119/ROP2中,经PCR和酶切鉴定正确的重组质粒pUC119/ROP2-P30再以SacⅠ/HindⅢ双酶切克隆至表达载体pET28b上,鉴定正确的重组质粒pET28b/ROP2-P30转化大肠埃希菌表达菌株BL21-Codon Plus(DE3)-RIL,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。结果从弓形虫RH株基因组DNA中扩增出700 bp P30基因片段,成功构建重组质粒pET28b/ROP2-P30,该质粒经PCR和酶切鉴定,与预期结果一致,并在大肠埃希菌中高效表达,产生相对分子质量(Mr)约为69 000的重组目的蛋白。结论弓形虫ROP2和P301融合基因克隆成功,并表达出预期的复合重组蛋白ROP2-P30。展开更多
应用PCR技术从刚地弓形虫(Toxoplasma gondii)RH株的基因组DNA中扩增编码棒状体蛋白ROP2(rhpotry protein 2)的部分基因,构建pGEX-KG-ROP2重组表达质粒,经酶切、PCR及DNA测序鉴定后,将阳性质粒转入E.coli BL21CodonPlus中,在IPTG诱导下...应用PCR技术从刚地弓形虫(Toxoplasma gondii)RH株的基因组DNA中扩增编码棒状体蛋白ROP2(rhpotry protein 2)的部分基因,构建pGEX-KG-ROP2重组表达质粒,经酶切、PCR及DNA测序鉴定后,将阳性质粒转入E.coli BL21CodonPlus中,在IPTG诱导下表达,表达产物用SDS-PAGE和Western-blot分析鉴定。结果表明,扩增的ROP2基因与GenBank上发表的相应基因序列的同源性达99.9%,该基因可以在大肠杆菌中高效表达,表达的ROP2融合蛋白表观相对分子质量约为64000,可被兔抗弓形虫免疫血清识别。展开更多
文摘According to the published gene sequence of the major surface antigen (P30) of Toxoplasma gondii, a pair of primers were designed and synthesized. Using polymerase chain reaction (PCR), the coding sequences of P30 gene were amplified from a Chinese strain of T. gondii, The amplified gene fragment and plasmid pB220 were digested with EcoRI and BamHI and then ligated. The inserted gene fragment was sequenced by the chain termination method, the reading reveals that nucleotide sequence determined was the same as the P30 sequecne of RH strain pubilished by Burg (1988), except that one base was changed. The recombinant plasmid containing P30 gene was transformed to E. coli DH5α.After temperature inducing culture, the total cellular proteins were analysed by SDS-PAGE and Western blot. The results show that the p30 gene cloned into the plasmid could express in E. coli, and the expression product had immunogenicity.
文摘目的进行弓形虫棒状体蛋白2(ROP2)和膜表面蛋白1(P30)融合基因的克隆与表达,为弓形虫ROP2-P30基因工程复合抗原的制备做准备。方法半套式PCR扩增编码弓形虫P30的基因片段,克隆至已构建成功的重组质粒pUC119/ROP2中,经PCR和酶切鉴定正确的重组质粒pUC119/ROP2-P30再以SacⅠ/HindⅢ双酶切克隆至表达载体pET28b上,鉴定正确的重组质粒pET28b/ROP2-P30转化大肠埃希菌表达菌株BL21-Codon Plus(DE3)-RIL,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。结果从弓形虫RH株基因组DNA中扩增出700 bp P30基因片段,成功构建重组质粒pET28b/ROP2-P30,该质粒经PCR和酶切鉴定,与预期结果一致,并在大肠埃希菌中高效表达,产生相对分子质量(Mr)约为69 000的重组目的蛋白。结论弓形虫ROP2和P301融合基因克隆成功,并表达出预期的复合重组蛋白ROP2-P30。
文摘应用PCR技术从刚地弓形虫(Toxoplasma gondii)RH株的基因组DNA中扩增编码棒状体蛋白ROP2(rhpotry protein 2)的部分基因,构建pGEX-KG-ROP2重组表达质粒,经酶切、PCR及DNA测序鉴定后,将阳性质粒转入E.coli BL21CodonPlus中,在IPTG诱导下表达,表达产物用SDS-PAGE和Western-blot分析鉴定。结果表明,扩增的ROP2基因与GenBank上发表的相应基因序列的同源性达99.9%,该基因可以在大肠杆菌中高效表达,表达的ROP2融合蛋白表观相对分子质量约为64000,可被兔抗弓形虫免疫血清识别。
