The Isolation And Fabrication Of E0 Genetic Prokaryotic Expression Vector Of Bovine Viral Diarrhea Virus Isolated From Ningxia

Bovine viral diarrhea mucosal disease (BVD/MD) is an infectious disease caused by bovine viral diarrhea disease virus (BVDV). The disease occurs mainly in cattle, calves are more susceptible, and also infect other animals. It is mainly caused by diarrhea,acute and chronic mucosal disease, persistent infection, immune tolerance, female abortion, stillbirth and other, causing serious economic losses to the cattle industry.In order to investigate the epidemiology and molecular epidemiology of bovine viral diarrhea mucosal disease from Ningxia, control of the disease prevalence in the Ningxia region. The test using ELISA method investigate BVDV infection and the prevalence from Ningxia. And on this basis track and detect molecular biology from seropositive cattle to obtain local BVDV isolatesThis study of 326 serum samples to sent to the laboratory test and 278 ear tissue samples, using BVDV antibody ELISA test kit for antibody detection of serum samples, use of ear tissue samples BVDV antigen ELISA test kit is used to detect the antigen. The sample of BVDV serological studies. Test results showed that 326 serum samples of BVDV antibody positive rate was 90.2%,278 samples of ear tissue of BVDV antigen positive rate was 0.3%. Test result makes clear the Ningxia region samples of BVDV antibody positive rate is very high, it may be because makes samples are with symptoms such as diarrhea, abortion.Using cell culture technology isolate virus from the diagnosis of antigen positive cattle, and successfully obtain bovine viral diarrhea virus isolates, named BVDV-NX. The isolation strains on the MDBK cell proliferation culture, the cell appears plaque, Seine and fall off, and a series of typical cell lesions (CPE). Then collect venom, extracting total RNA virus and obtained BVDV-NX strains cDNA by the reverse transcription.5’-UTR gene fragments were amplified and sequenced by polymerase chain reaction (PCR) method. Using MEGA6 analysis software,choosing N-J (Neighbor-Joining) method to draw the phylogenetic tree.The test results showed that this strain of BVDV-NX belongs to BVDV genotype lb.According to published BVDV EO gene sequences on the NCBI design a pair of specific primers, using polymerase chain reaction (PCR) amplification of 710 bp EO genes,sequencing and sequence analysis.The PCR amplification of EO gene cloning into the prokaryotic expression vector of pET-32a, transformed into e. coli BL21 (DE3) to identify the double enzyme,construct prokaryotic expression vector pET-32a-E0,lay the foundation for further test...