The Study On The Cloning And Expression Of GB Gene Of Infectious Laryngotracheitis Virus

Avian infectious laryngotracheitis(ILT) is a world-wide-occurring and severe respiratory disease of chickens, which characterized by signs of respiratory depression, gasping, expectoration of bloody mucus, and high mortality. The causative agent was designated infectious laryngotracheitis virus (ILTV), or gallid herpesvirus 1, and classified as a member of the Alphaherpesvirinae subfamily of the Herpesviridae. For prevention, chickens are immunized with conventionally attenuated live vaccines. Most of these genetically uncharacterized virus strains are still moderately pathogenic, and they bear the risk of spontaneous reversion to a more virulent phenotype.As a prerequisite for the development of genetically engineered vaccines, most of the ILTV genome has been characterized by DNA sequencing over the last few years. These studies confirmed that ILTV possesses a herpesvirus type D genome. Research showed that the glycoprotein gB of is a major protective antigen. It can make body generating the antibody to eliminate infection. So gaining gB gene and its expressing product is key to the preparation of a new type vaccine. The expression of gB gene provided the basis on the preparation of vaccine and a new method to prevent chicks from ILTV infection.According to the published nucleotide suquences of gB gene of ILTV, a pair of primers used to amplify gB gene were designed and synthesized . The complete gB gene of a domestic isolation stain were amplified by PCR, and obtained about 2.6 kb fragment. A recombinant plasmid was constructed by cloning PCR product into pGEM-T vector. The sequence analysis showed that the nucleotide sequence consists of 2622 bp, encoding 874 amino acids. Comparing the isolate virus with Thome V882(England), ILTV 632 (US),and SA 2(Australia), we found that their nucleotide sequences homologies were 100%, 100% and 99.4% respectively, their amino acids sequences homologies were 99.8%, 99.9% and 98.7% respectively. The sequence comparison indicated that the gB gene of ILTV strain shares high homologies with those of strain Throne V882, strain 632 and a vaccine strain SA 2.The recombinant plasmid pGEM-T-gB was digested with EcoR I/BamH I, and the resulting fragment was inserted into the EcoR I/BamH I site of the pBV221, which was the multiple cloning sites of the vector.' The recombinant plasmids were selected and identified byrestriction enzyme digestion which indicate a prokaryotic expression plasmid pBV-gB was constructed successfully. E.coli component host DH5 a was transformed with the expression plasmid. The recombinant bacterial stain could express the gB glycoprotein after induced by temperature, and the recombinant proteins were analyzed on 12% SDS-PAGE, and detected by ELISA with anti-ILTV serum which was prepared iii SPF chick immumized with live vaccine. The recombinant bacterial stain DH5 a (pBV-gB) can partly protect SPF chickens from a lethal dose(aEID50)of ILTV.According to the nucleotide suquences of gB gene of ILTV and the restriction enzyme digestion of the shuttle vector, two pairs of primers used to amplify gB gene were designed and synthesized. Firstly, a recombinant plasmid was constructed by cloning PCR product into pY-a vector. Then the shuttle expressive vector system was constructed by cloning the hsp- a -gB gene into the downstream sequences of pRR3 vector. The recombinant plasmid was identified by restriction enzyme digestion and electrophoreted into M.smegmatis me2 155. At last the recombinant proteins were detected successfully by ELISA and western blot, which seems to be immunogenic crucially. The recombinant bacterial stain M.smegmatis me2 155 (pRR-a -gB ) can protect SPF chickens from a lethal dose (a EID50) of ILTV. As the current commercial ILTV vaccines have several disadvantages as mentioned above, the recombinant bacterine M.smegmatis me2 155 (pRR-a -gB) can be considered very safe and therefore a promising substitution of live attenuated ILTV vaccines for future eradication of avian infectious laryngotracheitis in combination with a differential dia...