牛源性无乳链球菌分离鉴定及cfb基因的克隆和真核表达载体的构建

Construction of Eukaryotic Expression Vector of cfb Gene from Bovine Streptococcus agalactiaee

为研制奶牛乳房炎无乳链球菌的基因工程疫苗,采集隐性奶牛乳房炎奶样,利用THB(Todd-Hewitt Broth)固体选择培养基和色素试验,并结合无乳链球菌种属特异性基因cfb,采用PCR技术从隐性乳房炎奶样中分离和鉴定无乳链球菌。根据GenBank已报道的链球菌cfb基因序列,经ORF分析和B细胞表位预测,利用Primer 5.0软件设计cfb因子富含B细胞表位区域引物,添加酶切位点和真核表达元件,以分离株无乳链球菌的基因组为模板,PCR扩增cfb基因并连接T载体克隆测序,经测序确定无误后,连接pcDNATM3.1 V5-His A(pcDNA-cfb)真核载体,转化DH5α感受态细胞,提取重组质粒进行酶切和测序鉴定。结果显示,从89份隐性奶牛乳房炎奶样中分离得到8株无乳链球菌,成功构建了真核表达载体(pcDNA-cfb)。THB和色素试验初步筛选,可以作为快速分离无乳链球菌的一种方法,利用Bcepred和Lasergene-Protean软件对分离得到的无乳链球菌cfb基因序列分析,在所克隆的cfb基因序列内有多个优势抗原表位,因此,有望作为无乳链球菌基因工程疫苗研制的抗原靶标基因序列,为进一步研究其基因工程疫苗提供基础条件。

In order to develop genetic engineering subunit vaccine,milk samples of cows with subclinic mastitis were collected. THB（ Todd-Hewitt Broth） solid selective medium and pigment test were adopted,combined with species specific gene cfb to isolate and identify Streptococcus agalactiae. ORF and B cell epitope analysis were carried out according to the cfb gene sequences published in GenBank. Primers designed with Primer 5 in cfb＇s B cell epitope enriched sequence and eukaryotic expression elements and restriction enzyme sites were added. Genome DNA of the isolates were extracted and served as PCR templates for cfb amplification,prior to link with the T-A cloning vector for sequencing. Correctly cloned cfb sequence were then linked to the pcDNATM3. 1 V5-His A（ pcDNAcfb） and transformed to DH5α competent cell for plasmid proliferation and sequencing. Results showed 8 strains of Streptococcus agalactiaee were characterized from 89 milk samples,and the pcDNA-cfb was successfully constructed.Primary selection by THB selective medium and pigment test could be used for quick isolation of Streptococcus agalactiae. Analysis of the cfb sequence by Bcepred and Lasergene-Protean software showed the cloned cfb sequence contained multiple dominant B cell epitope,thus had the potential to serve as target gene sequence for genetic engineering subunit vaccine of Streptococcus agalactiae. This work could served as the basis for further developing genetic engineering subunit vaccine for Streptococcus agalactiae.