Expression And The Establishment Of The Indirect ELISA Of Pseudo Rabies Virus GE Protein

The pathogen of Pseudo rabies (PR) is Pseudo rabies Virus (PRV), which belongs to the herpesvirus subfamily of the herpesvirus. It can be infected by many mammals, which will lead death to cats, dogs and sheep. PR in pigs is an infectious disease by direct or indirect contaction. As the natural host of PRV virus, pig can excrete virus all through its life, which causes serious economic losses. The genome of PRV is approximately 150 kb, the GC content is 74%, which can encode for more than 70 species of viral proteins. As an important virulence of PRV and non-essential glycoprotein, glycoprotein E (gE) plays critical roles in PRV infection, knocking out of gE gene can obtain gene weaken vaccine. In China, pig farms being infected by PRV exhibited high gE antibody positive rate increasing, the sow stillbirth and piglets showed neurological symptoms and death, etc. After outbreak of PRV, PRV can also be isolated from the died piglets, which had be immuned with PRV gE gene deletion vaccine. This shows that only inoculation of gE gene deletion vaccine without using gE protein antibody diagnosis technology is unable to control the PR disease effectively. Because gE were expressed in all wild strain, it can be used as the protein marker between vaccine strains and wild strains. Therefore, it is necessary to establish a rapid diagnostic method for gE-ELISA.Nowadays, the main clinical use of gE-ELISA kit is imported, which is expensive and not suitable for large-scale universal use. In order to establish a sensitive and specific serological method, we isolated the PRV wild-type strain HNNYV1 from Henan Nanyang, PCR amplification of main antigen epitope area of gE gene, about 318 bp, then it was subcloned to pET28a(+) expression vector and transformed into BL21 strain, the expression of recombinant gE protein was best induced in 37? 220 rpm with 0.5 mM IPTG after 8 h incubation. Because the recombinant protein have His tag, after affinity chromatography column purification, western blot showed that it has good antigenicity and specificity. The indirect gE-ELISA method was established to detect the wild-type antibody of pseudo rabies virus by optimizing the conditions of the indirect ELISA. Specific method was as follows:using the purified protein of gE as coating antigen, package amount is 0.5 ?g/mL,4% gelatin in 37? and being incubated by 2 h, influenza virus serum was 1:100 diluted, with 37? and 1.5 h incubation, enzyme standard antibody was 1:40000 diluted, with 37? and 1 h incubation, TMB chromogenic for 10 min, then OD450nm was tested, more than 0.463 was positive, less than 0.402 was negative, between judged to be suspicious, can repeat the experiment to determine. There is no cross reaction between porcine circovirus virus (PCV), PRRSV and PRV gB. The method and IDEXX gE- ELISA antibody detection kit of coincidence rate was 91.67%. This study provide an indirect gE ELISA detection method and a wild rapid diagnosis and epidemiological investigation for PRV infection.