Abstract: 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 pcDNA^TM 3.1 V5-His A(pcDNA-cfb)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.

Key words: Recessive bovine mastitis, Streptococcus agalactiaee, cfb gene, Eukaryotic expression vector

CLC Number: 

• Q78