Construction And Expression Of Eukaryotic Vector Of ESP Gene From Bovine Enterococcus

Bovine mastitis is one of the most serious diseases causing loss of cow industry.It also causes mammary glands losing function of producing milk, makes a lot of cow culled, and leads to economic losses in the cow industry.Many factors associate with bovine mastitis. Among them, the infection of microorganisms is revealed as the main reason.Enterococcus are commensals of the intestinal tract of humans and animals and major opportunistic pathogens. It can cause cutaneous infection, soft tissue infection,celiac infection, blood poisoning, endocarditis and encephalitis.Enterococcal mastitis was relatively uncommon compared with subclinical and clinical mastitis caused by major pathogens. The decrease of milk quality and udder health problems within herds caused by Enterococcus have been reported. The occurrence and prevalence of enterococcal mastitis threatens human health as well as public safety.Therefore, the study was conducted to develop new genetic vaccines against enterococcal mastitis. Based on the isolates of Enterococcus from milk samples of cows with clinic and subclinic mastitis, and make full use of molecular biology,genetic engineering and recombinant DNA technology, the research was carried out to investigate anti-enterococcal mastitis genetic engineering subunit vaccine.Firstly, 96-well plate broth microdilution method was adopted to test the susceptibility of isolates of Enterococcus to 11 antimicrobial agents according to the NCCLS standards, and calculated MIC to each antibiotic. The results could be candidates in clinic use.Secondly, enterococcus surface protein(esp) gene was amplified from Enterococcus by polymerase chain reaction(PCR), and inserted into p CR@2.1 Vector with T/A clone. Linear pc DNA3.1-His B and esp gene obtained by cleaving theeukaryote vector pc DNA3.1-His B and p CR@2.1 Vector with esp by Eco RI and Kpn I.Esp fragment and pc DNA3.1-His B Vector isolated were ligated with T4 ligase. The recombination eukaryote plasmid was transformed into Tran1-T1 competent cells.Positive colonies were picked and the plasmid was extracted from Tran1-T1 competent cells.Lastly, by liposome method, MCF-7 cells were transformed by esp gene, and cultured in vitro. With methods of Western blot 、 PCR, results showed that the recombinant plasmid successfully expressed their exogenous genes and the protein in vitro, and the eukaryote vector pc DNA3.1 His B-rib esp was successfully constructed.This study will provide breakthrough ideas, accumulate raw data, and establish the basis of technology for researches of new genetic vaccines against enterococcal mastitis. Furthermore, the results of this research will provide the basis of animal immunization and pre-clinical research of Enterococcus vaccine, and the adoption of a scientific and rational prevention and control measures to restrict the occurrence of bovine mastitis.