新疆大学生命科学与技术学院,生物资源与基因工程重点实验室
纸质出版:2013
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[1]杨春静,芦清霞,克力比努尔·热合曼,等.假结核耶尔森菌侵袭素蛋白基因的克隆及原核表达[J].新疆大学学报(自然科学版),2013,30(02):144-147.
杨春静, 芦清霞, 克力比努尔·热合曼, et al. 假结核耶尔森菌侵袭素蛋白基因的克隆及原核表达[J]. Journal of Xinjiang University (Natural Science Edition in Chinese and English), 2013, 30(2): 144-147.
目的克隆假结核耶尔森菌侵袭素蛋白基因并在原核表达系统中表达.方法根据GenBank上登录的假结核耶尔森菌侵袭素蛋白核酸序列设计引物
用PCR方法扩增假结核耶尔森菌侵袭素蛋白基因
并将其插入克隆载体pMD18-T中
测序鉴定正确后再将其克隆到原核表达载体pGEX-4T-1上
得到重组质粒pGEX-4T-1-inv.将重组质粒转化大肠杆菌BL21(DE3)感受态细胞
经IPTG诱导
SDS-PAGE电泳检测.结果假结核耶尔森菌提取的基因组
经PCR扩增
得到一段591 bp的片段
与预期结果一致;SDS-PAGE电泳分析
所得重组蛋白的分子量约46 kDa
与预期结果相同;重组菌体超声裂解后
经12%的SDS-PAGE电泳分析
结果显示该重组蛋白为包涵体蛋白.结论克隆的侵袭素基因核心片段可以在原核细胞中高效表达.
Objective To clone invasin gene from Yersinia pseudotuberculosi and express it in prokaryotic system.Methods According to the nucleic acid sequence of Yersinia pseudotuberculosi invasin gene deposited in GenBank
a pair of primers were designed and synthesized.The invasin gene was amplified by PCR and inserted into the pMD18-T vector
after confirmed with sequencing
the gene was subcloned into the prokaryotic expression vector pGEX-4T-1 and the recombinant plasmid pGEX-4T-inv was constructed.For expression of recombinant invasin protein
the recombinant plasmid was transformed into competent cell of E.coli BL21(DE3) after IPTG induction.The expressed fusion protein was confirmed by SDS-PAGE electrophoresis.Results An expected fragment 591 bp of invasin gene was amplified from the genomic DNA extracted from Yersinia pseudotuberculosis by PCR.The recombinant invasin gene was expressed after the induction with isopropyl-β-d-thiogalactoside(IPTG).The expressed GST-fused invasin core protein was stained on SDS-PAGE gel which showed the expected molecular weight of 46 kDa compared with the standard protein marker.After ultrasonic degradation
the recombinant was analyzed by 12% SDS-PAGE electrophoresis and the result showed that the recombinant protein was inclusion body.Conclusions Core fragment of the clonal invasin gene can highly express in prokaryocyte.
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王鑫,景怀琦.假结核耶尔森菌研究进展[J].中国人兽共患病学报,2007,23(10):1041-1045.
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Clark M,Hirst B,Jepson M.M-cell Surface Beta1Integrin Expression and Invasin-mediated Targeting of Yersinia pseu-dotuberculosis Tomouse Peyer’s Patch M Cells[J].J Infect Immun,1998,66(10):1237-1243.
Isberg R,Voorhis D,Falkow S.Identification of Invasin:a Protein That Allows Enteric Bacteria to Penetrate Cultured Mammalian Cells[J].J Cell,1987,52:769-778.
Leong J,Morrissey P,Marra A,Isberg R.An Aspartat Residue of the Yersinia invasin Protein Invasin Protein That Is Critical for Integrin Binding[J].J EMBO J,1995,14(5):422-431.
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