The Expression Of Baculovirus AcMNPV Gp64Gene In Cerevisiae Cells

Autograph californica multiple nucleopolyhedrovirus (AcMNPV) is a typicalrepresentative of baculoviridae, which can infect more than30lepidoptera insects.Baculovriuses are large, enveloped viruses with a double-stranded circular DNA genomeof134kb. In the infection cycle, AcMNPV produces two forms of virions, the occlusionderived virus (ODV) and the budded virus (BV). The Gp64protein is only found in theBV and the weight of molecular is64kD. Gp64protein is a class â…¢ viral membranefusion protein that exists at one end of the BV and it is mainly composed of trimerswhose structure is connected by disulfide bonds.The protein can be expressed duringboth early and late period of virus infection. The Gp64protein is one of key protein whenvirus gets into host cells. Gp64, the major envelope glycoprotein of AcMNPV BV, isimportant for host cell receptor binding and mediating low-pH-triggered membranefusion during endocytosis. Also Gp64is necessary for efficient budding and productionof infectious virions. In the present work, Gp64protein was expressed from theprokaryotic expression system and used to gain the Gp64protein antibody, which ishelpful to test the expression of Gp64gene in yeast expression system.1. Expression of AcMNPC Gp64gene in E.coli: In order to express the Gp64gene, a pairof primers was designed according to the genome sequence published on NCBI. TheGp64gene was amplified from the AcMNPV Bacmid by PCR using the primers with theEcoR I and Sal I sites, the products from PCR were tested for sequencing and cut by theEcoR I and Sal I and then cloned into a prokaryotic expression plasmid pET28a(+) thathas been cut by EcoR I and Xhol I enzyme. This recombinant plasmid is namedpET28a-Gp64, and it was identified with restrition enzyme digestion and sequenced. Theplasmid pET28a-Gp64was transformed into E.coli BL21and induced by IPTG. Theresult of SDS-PAGE indicated that pET28a-Gp64gene was not expressed inprokaryoptic expression system.2. Expression of the truncated AcMNPC Gp64gene in E.coli: In order to express theGp64gene, a pair of primers was designed according to the genome sequence publishedon NCBI and removed the TM domains. The truncated Gp64gene was amplified fromthe AcMNPV Bacmid by PCR. Then using the same method the truncated Gp64gene from PCR was inserted into the prokaryotic expression plasmid pET28a(+). Thisrecombinant plasmid is named pET28a-truncated-Gp64and it was identified withrestrition enzyme digestion and sequenced. The plasmid pET28a-truncated-Gp64wastransformed into E.coli BL21and induced by IPTG. The result of SDS-PAGE indicatedthat pET28a-truncated-Gp64gene was expressed in prokaryoptic expression system andthe purpose protein is about40kD. The conditions of protein expression were optimized,and high purity of target protein was obtained for by electrical dialysis. In the end, whiterabbit immune experiment was carried out to gain Gp64antibody using these purifiedprotein.3. Expression of the AcMNPC Gp64gene in cerevisiae cells: a pair of primers wasdesigned according to the genome sequence published on NCBI. The Gp64gene wasamplified from the AcMNPV Bacmid by PCR using the primers with the EcoR I and SalI sites and then cloned into a eukaryotic vector pGBKT7that has been cut by the sameenzyme. This recombinant plasmid is named pGBKT7-Gp64, and it was identified withrestrition enzyme digestion and sequenced. The plasmid pGBKT7-Gp64was transformedinto AH109and the expression in Yeast system was analyzed using SDS-PAGE andWestern Blotting analyseIn summary, in the present work baculovirus AcMNPV capsule membrane proteinGp64gene was expressed in E.coli expression system and Yeast expression systemrespectively. For further research of the virus infection, this work provides effectivedetection reagent for Gp64produced in the process of virus infection.