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犬肺表面活性蛋白A的克隆及其在昆虫杆状病毒系统的表达
熊海凤,吴汉文,黄学婷,李明,周倩,崔雅倩,祁克宗,刘红梅
0
(兽医病理生物学与疫病防控安徽省重点实验室,安徽省动物性食品质量与生物安全工程实验室,安徽农业大学动物科技学院,合肥 230036)
摘要:
利用RT-PCR方法扩增犬肺表面活性蛋白A(canine lung surfactant protein A, cSP-A)基因,将其连接至pMD18-T载体,经测序正确后,使用DNAMAN软件比对不同种属SP-A基因序列;将cSP-A基因片段经双酶切后连接至pFastbac1载体得到pFastbac1-cSP-A;再将其转化至DH10Bac感受态细胞,获得重组Bacmid质粒,然后转染sf9细胞得到P1代重组杆状病毒P1 rBv-cSP-A,P1 代病毒连续扩增3代后,取P4 rBv-cSP-A感染sf9细胞表达cSP-A重组蛋白,利用蛋白免疫印迹和间接免疫荧光试验鉴定表达产物。结果显示cSP-A基因大小为699 bp;不同种属间SP-A 的C末端碳水化合物识别结构域的同源性为49.1% ~95.5%;经Western Blot和IFA鉴定,重组cSP-A蛋白在细胞内表达未分泌到胞外,分子量大小约为31.84 kDa。
关键词:    肺表面活性蛋白A  克隆  昆虫杆状病毒表达系统  表达
DOI:10.13610/j.cnki.1672-352x.20220705.001
投稿时间:2021-11-25
基金项目:安徽农业大学研究生创新项目(2021yjs-20)和国家自然科学基金(31872445)共同资助。
Cloning of canine lung surfactant protein A and its expression in insect baculovirus system
XIONG Haifeng,WU Hanwen,HUANG Xueting,LI Ming,ZHOU Qian,CUI Yaqian,QI Kezong,LIU Hongmei
(Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, Anhui Province Engineering Laboratory for Animal Food Quality and Bio-safety, School of Animal Science and Technology, Anhui Agricultural University, Hefei 230036)
Abstract:
RT-PCR was used to amplify canine lung surfactant protein A (canine lung surfactant protein A, cSP-A) gene, which was ligated into pMD18-T vector. DNAMAN software was used to compare cSP-A gene sequences from different species after sequencing correctly. cSP-A gene was digested by double enzymes and then cloned to pFastbac1 vector to prepare pFastbac1-cSP-A, which was further transformed into DH10Bac competent cells to obtain recombinant bacmid plasmid, and transfected into sf9 cells to generate P1 recombinant baculovirus rBv-cSP-A subsequently; P1 viral stock was propogated serially for three times. The recombinant protein was expressed in sf9 cells infected with P4 rBv-cSP-A, and the expressed product was identified by western Blot and indirect immunofluorescence test. The result showed that the length of cSP-A gene was about 699 bp. The homology of SP-A carbohydrate binding domain among different species was 49.1%-95.5%. Recombinant cSP-A protein was expressed in cells but not secreted out of cells, and its molecular weight was approximately 31.84 kDa by Western Blot and IFA.
Key words:  canine  lung surfactant protein A  cloning  insect baculovirus expression system  expression

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