Construction And Expression Of HA - VP2 Recombinant Baculovirus

Infectious bursal disease virus (Infectious bursal disease virus, IBDV) is acute and highly contagious disease pathogens which can cause disease in chicken and turkey. VP2 is the major protective antigen of host. VP2 concernes with the strength of IBDV virulence, antigenic variation and virus neutralizing antibody induction and identify. The major direction of the prevention and control of IBD research is exploiting the expression vector to express VP2 used to prepare genetically engineered vaccine. Baculovirus expression vector system (Baculovirus Expression Vector System, BEVS) whose foreign genes expression vector is a baculovirus, and whose receptor is insect cell, is a safe, efficient vector system with large capacity. It is easy to screen recombinant virus, and the recombinant protein could be folded and modified with biological activity and so on. Epitope tagging (epitope tag) is the attached epitope fuse to detect protein so that can be used to detect the expression of protein, and also can be used to purify the target protein by affinity chromatography or to locate subcellular proteins by immunofluorescence. HA-tag is the more widely used epitope tagging. This issue fused HA-TAG and IBDV-VP2, and used Bac-to-Bac baculovirus expression vector system to express the fusion protein in CHO cell lines. Compared and analyzed the influence of the level of protein expression in different recombinant vector, then chose the preferred expression system. This issue provides new ideas for preparing the genetic engineering of poultry vaccine.Using DNAstar software to design a pair of specific primers of the VP2 that refer to published IBDV-VP2 sequence on GenBank, and fused the HA-TAG gene to the IBDV-VP2 gene sequence of the 5’end. HA-VP2 gene was amplified by PCR, and cloned into pMD18-T vector. Recombinant plasmid named pTZF-HA-VP2 containing the VP2 gene was gained by the way of restriction enzyme analysis, bacteria PCR and nucleotide sequencing identified. Then HA-VP2 gene was subcloned into the previous transfer vector pWK, pWK-I, pLM, pLM-I, pSD, pSD-I, pWKC, pLMC, pSDC, which had been constructed. By different combination enzyme digestion it was proved that the recombinant transfer vectors with different expression cassettes which named pTZF-WK-HA-VP2, pTZF-WK-I-HA-VP2, pTZF-LM-HA-VP2, pTZF-LM-I-HA-VP2, pTZF-SD-HA-VP2, pTZF-SD-I-HA-VP2, pTZF-WKC-HA-VP2, pTZF-LMC-HA-VP2, pTZF-SDC-HA-VP2 was gained. These transfer vectors were transformed into competent cells of E.coli DH10Bac, and formed recombinant shuttle plasmids rBac-TZF-WK-HA-VP2, rBac-TZF-WK-I-HA-VP2, rBac-TZF-SD-HA-VP2, rBac-TZF-SD-I-HA-VP2, rBac-TZF-LM-HA-VP2, rBac-TZF-LM-I-HA-VP2, rBac-TZF-WKC-HA-VP2, rBac-TZF-SDC-HA-VP2, rBac-TZF-LMC-HA-VP2 by Bac-to-Bac Baculovirus expression vector system. These plasmids transfected sf9 insect cells and gained the recombinant baculovirus named P1, continuing to proliferate the virus to P3 generation. Titers of the virus were 1-2×108 pfu/mL, analyzed by lacuna. Finally, the received nine viruses infected CHO cells in the same time, then used SDS-PAGE and Western-blotting to detect the expression of VP2 protein.The results show that the optimized baculovirus expression vector system expressed VP2 with antigenicity of infectious bursal disease virus. It laid a solid foundation for exploring the modified baculovirus as a gene delivery vector in the development of genetic engineering of poultry vaccine.