The Study On High-level Expression Of Hepatitis B Virus Surface Antigen (PreS2+S) And Immunnogenicity Of Products Expressed In Silkworm/BmNPV Expression Vector System

(一)Foreign genes can be expressed effectively at low cost in silkworm-BmNPV expression vector system (Silkworm Bioreactor),which is widely noticed in the world. Numerous useful proteins have been expressed and the expression levels were higher in silkworm-BmNPV expression vector than that of others systems. However,the quantities of foreign gene products are far lower than that of the original polyhedra. In this study,we explored functional mechanism of polyhedron gene sequence on expression level of HBsAg(PreS2+S) genes and optimized the conditions for high-level expression in Silkworm-BmNPV expression vector system.A 12 bp sequence of the 5' end from the polyhedrin protein gene of BmNPV was ligated to the 5'end of HBsAg( PreS2+S ) protein coding sequence by PCR. The fusion product coding for HBV surface antigen medium sized (HBMp) with 4-additional AA of BmNPV polyhedrin protein was obtained. The recombinant transfer vector pBacPAK-HBMp was constructed by insertion of the HBMp coding sequences into the multiple cloning site of transfer vector pBacPAK.8. BmN cell line was co-transfected with pBacPAK-HBMp plasmid and linearized baculovirus BacPAK6 DNA by Dosper agent. The homology of recombinant virus BmPAK-HBMp was obtained and identified by plaque assay and baculovirus contains the HBMp gene was confirmed through PCR and DNA dot blotting.The expressed rHBsAg was determined by ELISA after infecting Bm-N cells and pupae with recombinant virus BmPAK-HBMp and BmPAK-HBM (containing nonfusion HBV surface antigen medium sized). The results showed that expression amount of rHBsAg infected with BmPAK-HBMp reached 3.98ug/2xl06cells and 46.6ug/mL in BmN cells and in pupa hemolymph respectively. On contrary,expression amount ofrHBsAg reached 2.58ug/2106 cells and 26.3ug/mL respectively where has infected with BmPAK-HBM. Expression amount of rHBsAg in the former was increased 60% and 80% respectively than the latter. Antigenicity of PreS2 of fusion protein was increased 8 times and 10 times in BmN cells and in pupa hemolymph respectively than nonfusion HBsAg. The results also showed that kinetics of HBsAg and PreS2-Ag was different,the time of the high expression level of PreS2-Ag was earlier one or two days than that of HBsAg,and the efficiency increased of PreS2-Ag is about 13 times which is higher than that of HBsAg. These data implied that the expression of HBV medium protein (encoded by PreS2 gene and S gene) could be earlier then that of small protein (encoded only by S gene). The polyhedrin gene sequence in recombinant virus BmPAK-HBMp can be more benefit to enhancing the expression of the HBsAg(PreS2+S) from ATG initial codon at the 5' ends of PreS2 gene.Pupal hemolymph infected with recombinant virus BmPAK-HBMp and BmPAK-HBM showed four protein bands of about 31,'30,28 and 25 kD in the Western blotting profiles,due to the two ATG initial codons at the 5' ends of both PreS2 and S gene. This implied that the 31 kD initiated from ATG codon of PreS2 gene,the 30 kD encoded by the S gene was glycosylated form of S protein,the 25 kD initiated from ATG codon of the PreS2 gene was disassembled form of 31 kD and the 28 kD was a glycosylated form of25kD.Under optimal condition the expression level of foreign gene was considerably affected on hosts (silkworm race),the highest difference of expression level in various races can reach up to 8 times. Zhelei Chunxiao,Feng 1 54A and Baiyu Xinhang are the races of choice for expression of HBsAg (PreS2+S). The temperature at which the pupae were kept after treatment was greatly affected the expression level of foreign gene. Expression level in pupa is 32 times higher at 23C -26C than that at 30C,and decreased by 50% at 20C,The level of expression was influenced to some extent by the titre of virus used for infection and rearing season. Higher level of expression of HBsAg can be obtained from pupa infected with higher virus titre. The expression level was 1 time higher inspring than that of autumn. The expression level was decreased little in coco...