| Bovine viral diarrhea is a very complicated and important infection caused by bovine viral diarrhea virus (BVDV),it has a variety of clinical symptoms. The main features of the disease are fever, cough, mucosal erosion, ulcer, leukopenia, diarrhea, and persistent infection, immune tolerance, immune suppression, congenital defects, pregnant cows abortion, stillborn fetus or fetal malformation.It is worldwidle distribution,and it is reported that there are more than20provinces which BVDV exist in our country.BVDV can infect all ages of the cattle,symptom of persistent infection individual is hiding,the rate of animal carried virus is high, especially BVDV affect infection animal breeding seriously, BVDV become invisible killer for farm benefit,and brings huge economic losses to the livestock industry.Bovine rhinotracheitis is composed of infectious bovine rhinotracheitis virus (IBRV) which also known as bovine herpes virus type1in cattle and wild cattle and it caused an acute, febrile, contagious disease. The main features are fever, difficulty breathing, rhinitis, sinusitis and upper respiratory tract inflammation,it also can cause abortion and stillbirth, enteritis and cow calf encephalitis.At present, there are various cattle in this virus infection in most of the provinces in China, while the absence of immune protection measures properly, the disease is on the rise,it caused serious harm to the cattle industry for greatly effects to cattle fattening rate/milk production and reproductive.At present,the trend of these two kinds of disease infection increased significantly,they are a serious challenge to China’s large-scale cattle industry. However, the aspects of these two kinds of disease diagnosis and treatment of drug development is still very weak in our country.Therefore, at present,the study on development of BVDV/IBRV diagnosis and vaccine is an important task, and it has great significance to these two kinds of disease prevention and control.BVDV E2protein is the major glycoprotein, the outer surface of the N-terminus projecting BVDV envelope is the main part of the decision of BVDV antigen and also involved in the anti-BVDV antibody mediated immune response and host-cell recognition and adsorption.IBRV encoding11kinds of glycoprotein,wherein the gD protein is the major glycoprotein of IBRV,it is the main molecular on virus particles and when virus infected,it is required in virus adsorption and invade the host cells which can be induced high levels on cellular immunity and humoral immunity by gD protein,and it can cause high and persistent cell immunity. Antibody anti gD is the best to neutralize the virus effect.These results suggest that E2and gD proteins are preferred antigenic specificity of BVDV and IBRV.Based on this,this paper firstly uses the PCR method to obtain BVDV E2and IBRV gD gene respectively.PEASY-E2and pEASY-gD recombinant vectors were constructed by pEASY-T3clone technology.And then cloned into pET28a vector to construct pET28a-E2-gD) recombinant vector.the PCR, restriction enzyme digestion and sequencing correct recombinant vector was transformed into E.coli BL21(DE3).37℃,protein expression of IPTG induced by1mM,Purification of recombinant protein affinity chromatography, purified protein concentration is2.69mg/ml by BCA kits.the Western-blot results show that it has a specific immunogen.Immunization of rabbits to prepare antiserum, results of serum neutralization test showed that Neutralizing antibody titer was44.7(IBRV)/22(BVDV).The results showed that, E2-gD protein better immunogenicity, which can induce high titers of neutralizing antibody, and lay the foundation for the development of diagnostic antigen BVDV/IBRV two virus and subunit vaccine. |
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