Cloning Of Cysteine Protease Gene From BmNPV And Expression In Silkworm Bioreactor

Baculoviruses are widely used for expressing heterologous proteins, ininsect cell culture or in insect larvae. The baculovirus expression system hasbecome a vital expression tool for the production and characterization of alarge variety of recombinant proteins. For improve the frequency of silkwormbaculovirus expression vector system (BEVS) and the efficiency ofbaculovirus biopesticides, in this study, we cloned the cysteine protease genefrom BmNPV genome DNA by amplified PCR and under the control of thepolyhedrin promoter, the cp gene was successfully expressed in the BEVS. A gene encodes a papain type cysteine protease with cathepsin L-likecharacteristics was obtained from the BamHI F fragrnent of the genome ofBmNPV-ZJ8 and cloned into the vector, pluscript-SK, using DNArecombinant technology. Sequence analysis revealed a 972 bp open readingframe and 323 amino acids long. Compared with the cp gene from othernuclear polyhedrosis viruses, it is high to the identity at the level of DNA. Of36 residues conserved among cathepsins B, H, L and S and papain, 31 wereidentical in BmNPV-CP. The phylogenetic tree of cp gene was constructedaccording to the sequences of the reported cysteine protease genes, it is shownthat the cp gene was on the same branch with papain upper family. It revealsthe highest identity to the BmNPV-T3 and AcNPV, and it is in the differentgroup compared with other cp gene come from HaNPV, HzsNPV, SeNPV andCpGV, respectively.After sequence identified, for the construction of the recombinant virus,the cp gene was linked into the downstream of the strong polyhedrin promoterin the transfer vector, pVL-1393. According to the analysis of restrictionendonuclease digestion and agrose electrophoresis, cp has been successfullyinserted into the vector in the right direction. Subsequently, we incorporated cp gene into the prokaryotic expressionvector pET-28a and expressed in E. coli BL21. As we all known that E. coliexpress system is a kind of prokaryotic system, which short of the capacity ofpost-translational modification as comparison with Eukaryotic express system.Furthermore, the product of cp gene may plays a critical role in theliquefaction of host tissues, that is to say, the product of expression is harmfulto the growth of host and block the expression of itself. We contrasted thecourse times of E. coli growth and revealed there is something that restrainedthe growth of E. coli induced by IPTG, which hold recombinant plasmidpET-CP. So we mediated proved the function of cp inserted into the vector. Based on the culture of insect cells and used the technology oflinerization of virus genomic DNA, the recombinant transfer vector PVL-CPand parental virus (Bm-BacPAK6) DNA which has been cleaved were made toco-transfect Bm-5 cells with lipofectine. We found the infection efficiency ofrecombinant virus is lower and the time of cell degradation is longer than thecells infected with parental virus. Stable recombinant viruses were isolated andpurified by plaque assay and limiting dilution method.The newly molted 5 instar larvae and early puoae of silkworm Bombyxmori were infected with purified recombinant virus. PCR assay showed thatcysteine protease gene of baculovirus was expressed after 4-5 days p. i. Wegathered the hemolymph of infected silkworm and investigated the activity ofCP, in the light of the data, we gained the courses time of CP and revealed theyield of express product increased rapidly after injected 96 h and achieved itspeak after 120 hours p.i., then, the susceptible larvae began to liquefection anddie. The same phenomena occurred in puoae after 132 h. With SDS-PAGEassay, we found that between the molecular weight of 20-30 kD, there is aspecific band instead of the control sample, we thinked it is one of theproducts of cp. Because the abundence of 30 kD proteins in the hemolymph oflarvae is very high...