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

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。

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

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.