绵羊γ干扰素（IFN-γ）基因的克隆、表达与抗病毒活性初步研究
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摘要
根据C.J.Mclnnes等于1990年发表的绵羊IFN-γ基因序列设计并合成引物,以经ConA诱导的绵羊(湖羊)脾脏淋巴细胞提取的总RNA为模板,用RT-PCR方法扩增出长度为554bp的目的基因片段。将该基因克隆到pMD18-T载体上,经PCR、酶切鉴定和序列测定,结果表明获得了绵羊IFN-γ基因的克隆。该基因包含绵羊IFN-γ基因的全长为501个碱基的开放阅读框,编码166个氨基酸组成的功能蛋白。克隆到的绵羊IFN-γ基因与GenBank上已公布的人、牛、山羊、马鹿、马、骆驼、猪、狗、猫和鸡的IFN-γ核苷酸序列进行比较,其同源性分别为75.2﹪、96.6﹪、99.0﹪、95.8﹪、83.0﹪、88.8﹪、86.6﹪、82.4﹪、53.9﹪、32.9﹪;氨基酸序列同源性分别为61.7﹪、94.6﹪、98.2﹪、92.2﹪、77.8﹪、86.8﹪、77.8﹪、76.0﹪、39.5﹪、6.7﹪。与已发表的其它品种绵羊相比较,核苷酸和氨基酸序列同源性都为100%。
将绵羊IFN-γ基因亚克隆,插入到原核表达载体pPROEXHTa中,构建成绵羊IFN-γ基因原核表达重组质粒pPROEXHTa-OvIFN-γ。经PCR、双酶切鉴定,表明所构建的重组质粒为阳性。将该重组质粒转化大肠杆菌BL21(DE3),阳性菌落筛选后,以IPTG为诱导剂进行诱导表达,经SDS-PAGE和Western blot分析,结果显示,在分子量约为22.6 kDa处有明显的蛋白质表达条带,与预期的融合表达蛋白大小一致。表达的融合蛋白主要以不溶性包涵体形式存在,约占菌体总蛋白的48%。说明基因工程菌株BL21(DE3)/pPROEXHTa-OvIFN-γ能够高效表达绵羊IFN-γ融合蛋白。通过对绵羊IFN-γ原核表达产物的提取、纯化和复性,获得了活性蛋白。采用细胞
病变抑制法测定了复性蛋白抗牛病毒性腹泻病毒(BVDV)活性。结果显示,复性蛋白具有抗BVDV活性,抗BVDV活性为1.0×107U/mL。
According to the sequence of Ovine IFN-γgene published by C.J.Mclnnes in 1990, A pair of primers was designed to amplify Ovine interferon gamma gene by RT-PCR with Primer 5.0 software. The product of PT-PCR named OvIFN-γis approximate 554bp in length that amplified from the total RNA of lymphoid cell induced by Con A. The OvIFN-γgene was cloned into the pMD18-T vector and the recombinant plasmid was named pMD18-T-OvIFN-γ. Identifications of restriction enzyme, PCR and sequencing indicated that the OvIFN-γgene has been cloned successfully. The gene includes a complete open reading frame which is 501bp in length and encoding a functional protein that consists of 166 Amino acids. Compared with the published IFN-γgene sequence, the homology of nucleotide sequence was 75.2﹪with Human,96.6﹪with Bovine,99.0﹪with Goat,95.8﹪with red deer,83.0﹪with horse,88.8﹪with camel,86.6﹪with pig,82.4﹪with dog,53.9﹪with cat and 32.9﹪with chicken;the homology of amino acid sequence was 61.7﹪with Human,94.6﹪with Bovine,98.2﹪with Goat,92.2﹪with red deer,77.8﹪with horse,86.8﹪with camel,77.8﹪with pig,76.0﹪with dog,39.5﹪with cat and 6.7﹪with chicken.
OvIFN-γwas inserted in multiple cloning sites of the prokaryotic expression vector pPROEXHTa, The recombinant plasmid pPROEXHTa-OvIFN-γwas identified by restriction enzyme and PCR. Purified recombinant plasmid pPROEXHTa-OvIFN-γwere transformed to E.coli BL21(DE3),and then harvested in 37℃. The Recombinant engineering strain of BL21(DE3)/ pPROEXHTa -OvIFN-γwas induced by the IPTG.. SDS-PAGE and Western blot analyses showed the OvIFN-γgene has been highly expressed in E.coli BL21. The molecular weight of Expressed fusion protein is approximately 22.6kDa, the fusion protein exists in the form of inclusion body and the content is approximately 48 percent in total protein of BL21.
The results of purification of fusion protein indicated the active protein has been obtained by dialysis. Test of antiviral activity for the recombinant OvIFN-γpurified was done with the method of inhibiting CPE on MDBK by BVDV. Results of detection showed purified protein have activity against the BVD virus. The antiviral activity of recombinant OvIFN-γfor BVDV is 1.0×107U/mL.
将绵羊IFN-γ基因亚克隆,插入到原核表达载体pPROEXHTa中,构建成绵羊IFN-γ基因原核表达重组质粒pPROEXHTa-OvIFN-γ。经PCR、双酶切鉴定,表明所构建的重组质粒为阳性。将该重组质粒转化大肠杆菌BL21(DE3),阳性菌落筛选后,以IPTG为诱导剂进行诱导表达,经SDS-PAGE和Western blot分析,结果显示,在分子量约为22.6 kDa处有明显的蛋白质表达条带,与预期的融合表达蛋白大小一致。表达的融合蛋白主要以不溶性包涵体形式存在,约占菌体总蛋白的48%。说明基因工程菌株BL21(DE3)/pPROEXHTa-OvIFN-γ能够高效表达绵羊IFN-γ融合蛋白。通过对绵羊IFN-γ原核表达产物的提取、纯化和复性,获得了活性蛋白。采用细胞
病变抑制法测定了复性蛋白抗牛病毒性腹泻病毒(BVDV)活性。结果显示,复性蛋白具有抗BVDV活性,抗BVDV活性为1.0×107U/mL。
According to the sequence of Ovine IFN-γgene published by C.J.Mclnnes in 1990, A pair of primers was designed to amplify Ovine interferon gamma gene by RT-PCR with Primer 5.0 software. The product of PT-PCR named OvIFN-γis approximate 554bp in length that amplified from the total RNA of lymphoid cell induced by Con A. The OvIFN-γgene was cloned into the pMD18-T vector and the recombinant plasmid was named pMD18-T-OvIFN-γ. Identifications of restriction enzyme, PCR and sequencing indicated that the OvIFN-γgene has been cloned successfully. The gene includes a complete open reading frame which is 501bp in length and encoding a functional protein that consists of 166 Amino acids. Compared with the published IFN-γgene sequence, the homology of nucleotide sequence was 75.2﹪with Human,96.6﹪with Bovine,99.0﹪with Goat,95.8﹪with red deer,83.0﹪with horse,88.8﹪with camel,86.6﹪with pig,82.4﹪with dog,53.9﹪with cat and 32.9﹪with chicken;the homology of amino acid sequence was 61.7﹪with Human,94.6﹪with Bovine,98.2﹪with Goat,92.2﹪with red deer,77.8﹪with horse,86.8﹪with camel,77.8﹪with pig,76.0﹪with dog,39.5﹪with cat and 6.7﹪with chicken.
OvIFN-γwas inserted in multiple cloning sites of the prokaryotic expression vector pPROEXHTa, The recombinant plasmid pPROEXHTa-OvIFN-γwas identified by restriction enzyme and PCR. Purified recombinant plasmid pPROEXHTa-OvIFN-γwere transformed to E.coli BL21(DE3),and then harvested in 37℃. The Recombinant engineering strain of BL21(DE3)/ pPROEXHTa -OvIFN-γwas induced by the IPTG.. SDS-PAGE and Western blot analyses showed the OvIFN-γgene has been highly expressed in E.coli BL21. The molecular weight of Expressed fusion protein is approximately 22.6kDa, the fusion protein exists in the form of inclusion body and the content is approximately 48 percent in total protein of BL21.
The results of purification of fusion protein indicated the active protein has been obtained by dialysis. Test of antiviral activity for the recombinant OvIFN-γpurified was done with the method of inhibiting CPE on MDBK by BVDV. Results of detection showed purified protein have activity against the BVD virus. The antiviral activity of recombinant OvIFN-γfor BVDV is 1.0×107U/mL.
引文
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