Isolation And Identification Of Four Pseudorabies Virus Strains, Cloning And Sequence Analysis, And Expression Of Envelope Glycoprotein E (gE) Gene

Specific primers to amplify gE gene of Pseudorabies Virus (PRV) were designed according to the published sequence of gE gene(accession number AY170318) of PRV strain minA and PCR method was successfully developed to detect PRV by using these primers under the optimized conditions of the test.The result of PCR detection for PRV in samples of several organs including brain from piglets with clinical signs of PRV infection indicated that ,of five samples,four were PRV positive only and one was both PRV and PCV 2 positive.It suggested the disease is very common in the pig flocks in Henan province. Four PRV positive samples without PCV2 tested by PCR were passaged on PK-15 cells and typical cytopathic effect(CPE) on cell culture was observed after serial passage. Four cell cultures were PRV positive only tested by PCR and the isolates were named as strains HNJZ,HNDF, HNTY and HNNY,respectively.Another two pairs of specific primers to amplify different segments of gE gene of PRV were designed according to the published sequence (accession number AY170318) and used in PCR amplification.The full length of gE gene from strain HNJZ and the main epitope segment of gE gene from four isolates above were obtained by PCR amplification. The PCR products were purified, and then cloned into PGM-T vector. Recombinant plasmids were demonstrated to contain the different segments of gE gene by restriction enzyme digestion and nucleotide sequencing. Comparison of nucleotide and deduced amino acid sequences between HNJZ strain and other domestic or foreign strains of PRV showed that their nucleotide homology rate reachs to 97.3%～99.9%, and amino acid sequence homology rate reaches 94.8%～99.8%. The result indicated that gE gene is conservative and that HNJZ strain is nearest to domestic strain minA and the overseas strain Yangshan. Deduced amino acid sequence of gE gene from PRV strain HNJZ showed 4 variations of amino acid outside its main epitope segment. Homology rate of nucleotide and deduced amino acid sequences for the four isolated strains in Henan are all above 96%. These results further indicated that gE main epitope gene is quite conservative.Based on the sequence of gE gene in strain HNJZ, a pair of primers were designed and utilized to amplify main epitope gene segment by PCR. Then the gene segment was cloned into expression vector pET-28a. The right recombinant plasmid(pET-28gE)was screened out and identified by PCR and restriction enzyme digestion. After being induced by IPTG, the recombinant protein was expressed. It is about 37KD, which consistented with anticipate result. Western blotting indicated that the recombinant protein has good immunoreactivity.