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  

Cloning of canine lung surfactant protein A and its expression in insect baculovirus system

作　　者：熊海凤 吴汉文 黄学婷 李明 周倩 崔雅倩 祁克宗[1] 刘红梅[1] 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)

机构地区：[1]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

出　　处：《安徽农业大学学报》2022年第3期449-453,共5页Journal of Anhui Agricultural University

基　　金：安徽农业大学研究生创新项目(2021yjs-20);国家自然科学基金(31872445)共同资助。

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p MD18-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。RT-PCR was used to amplify canine lung surfactant protein A(canine lung surfactant protein A, c SP-A)gene, which was ligated into pMD18-T vector. DNAMAN software was used to compare c SP-A gene sequences from different species after sequencing correctly. c SP-A gene was digested by double enzymes and then cloned to pFastbac1vector to prepare pFastbac1-c SP-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-c SP-A subsequently;P1 viral stock was propogated serially for three times. The recombinant protein was expressed in sf9 cells infected with P4 rBv-c SP-A, and the expressed product was identified by western Blot and indirect immunofluorescence test.The result showed that the length of c SP-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.

关 键 词：犬 肺表面活性蛋白A 克隆 昆虫杆状病毒表达系统 表达 

分 类 号：S858.292.53[农业科学—临床兽医学]