马驽巴贝斯虫新疆伊犁株Bc48基因克隆、表达及其多克隆抗体的制备

为表达马驽巴贝斯虫新疆伊犁株Bc48基因，制备多克隆抗体，试验根据GenBank中马驽巴贝斯虫Bc48基因序列，设计合成1对特异性引物，以提取的新疆株Bc48基因组DNA为模板进行PCR扩增，将扩增产物克隆至原核表达载体pET-28a（+）中，阳性质粒转化至大肠杆菌BL21（DE3）感受态细胞中，经IPTG诱导后表达产物进行SDS-PAGE分析，切胶纯化蛋白后免疫6周龄雌性BALB/c小鼠，制备多克隆抗体；同时把该基因克隆至真核表达载体pCMV-N-flag中，将阳性质粒转化至大肠杆菌DH5α感受态细胞中，提取pCMV-N-flag-Bc48质粒转染到293T细胞中进行真核表达。结果显示，获得了大小约为15 ku的融合蛋白，与预期目的蛋白大小相一致；免疫BALB/c小鼠制备的多克隆抗体经ELISA和Western blotting检测、验证，能特异地识别相应抗原，其抗体效价可达1：128 000，说明该多克隆抗体有较高的抗体效价和特异性；真核表达质粒在293T细胞中表达Bc48蛋白。本试验可为进一步研究马驽巴贝斯虫功能基因及建立快速的检测方法奠定基础，在虫株的分类学研究及候选疫苗的研制中有重要意义。

Cloning and Expression of Bc48 Gene of Babesia caballi Xinjiang Yili Strain and Preparation of Its Polyclonal Antibody

In order to express Bc48 gene of Babesia caballi from Xinjiang Yili,and prepare its polyclonal antibody,a pair of specific primers was designed according to Bc48 gene sequence of Babesia caballi in GenBank.The genomic DNA was extracted from Babesia caballi,and Bc48 gene was cloned into pET-28a(+) expression vector.The positive plasmid was transformed to Escherichia coli BL21(DE3).After IPTG induction,the product was analyzed by SDS-PAGE,six weeks old female BALB/c mice were immunized with purified Bc48 protein to prepare polyclonal antibodies.Meanwhile Bc48 gene was cloned into eukaryotic expression vector pCMV-N-flag.The positive plasmid was transformed to Escherichia coli DH5α,eukaryotic expression of pCMV-N-flag-Bc48 plasmid was transfected into 293T cells.The results showed that the recombinant protein with 15 ku was obtained,which was consistent with the expected protein size;Western blotting analysis showed that polyclonal antibodies could specifically identify the corresponded antigens;ELISA analysis showed that the antibodies had high titer (1:128 000);And eukaryotic expression plasmids expressed Bc48 protein in 293T cells.This test could lay the foundation for further study on the functional genes and establish quick detection method of Babesia caballi.It had important significance in the study of strain taxonomy and the development of candidate vaccine.