Development Of A Novel BmNPV Expression Vector System Using Silkworm As Bioreactor And The Application

Baculovirus has been proven to be the most powerful and versatile eukaryotic expression vectors available today. In this system, recombinant protein was expressed through the infection of insect or its culuture cells by using a recombinant baculovirus, which carrying the gene of interest. BEVS has lots of advantages including well high-level expression under control of strong promoter such as polyhedrin and p10 promoter, post-translational modifications, capacity of large gene insertions, as well as simultaneous expression of multiple genes. Compared with the mammalian expression, protein expressed by BEVS is safer to human being because the baculoviruses have very restricted host range.The most popular vector in BEVS is Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). AcMNPV expression system has been well developed in many fields and was widely applied, specially in American and European countries. Compared with the AcMNPV system, the BmNPV was not applied as widely as AcMNPV, however, the most attractive advantage of BmNPV expression is that BmNPV can infect silkworm larva or pupa, which can be easily reared. When expressed in the silkworm, the expression can uauslly reach a higher level over other systems, moreover, the production cost is much lower. In our country, silkworm is very important economic insect, and large amount of the larvae can be easily obtained, and thus it is very promising to use the silkworm as a "biofactory" to produce the recombinant protein in biotechnological industry.However, the BmNPV expression system has some bottlenecks in techniques, particularly in large-scale production. It includes:(1) The construction and purification of recombinant BmNPV is still laborious and time-taking. The traditional method for construction of recombinant BmNPV through homologous recombination in insect cells needs a few rounds of plaque assay. It needs skillful technique and usually takes 2-3 months; (2) In the late stage of infection, recombinant protein is usually degraded, which greatly decreases the protein production level. BEVS is a lytic expression system. There are several proteinase coded by the BmNPV itself. Along with the expression of foreign protein, the proteinases are also expressed, especially in the late stage of infection; (3) The efficiency of virus inoculation to the silkworm through subcutaneous injection is too low. Usually the polyhedrin gene in BmNPV was replaced by the foreign genes, so the recombinant BmNPV are polyhedrin negative virus. Without the protection of polyhedra, budded virus can hardly infect the silkworm by oral. The only way of recombinant BmNPV to infect silkworm larvae is through subcutaneous injection. For large-scale production, it is big problem.This study tried to solve the above problems in order to make the BmNPV system more efficient. The achievements were summaried as the following:[1] A novel BmNPV Bac-to-Bac/Bacmid expression system suitable for silkworm, Bombyx mori was successfully developed. According to the principle of AcMNPV Bac-to-Bac expression system, the BmNPV genomic DNA was similarly reconstructed. A fragment containing a kanamycin resistance gene, a mini F replication element and the LacZ-αgene fragment which harbors a mini-attTn7 element as the target site of transposon without disrupting the LacZ open reading frame was inserted to the polyhedra site of BmNPV and replaced the polh gene through homologous recombination. Thus a BmNPV shuttle vector BmBacmid was constructed. Further, a set of donor plasmids essential for this system was reconstructed, and these donor plasmids make the recombinant protein well secreted. Using the newly constructed BmNPV Bac-to-Bac/Bacmid expression system, the recombinant BmNPV with foreign gene can be rapidly generated through bacterial transposition in E.coli. [2] A novel BmNPV expression system which can produce the polyhedrin was constructed. Polh gene was cloned to the site of p10 gene and placed in the control of p10 promoter. Thus the Bmbacmid (polh+) DNA were constructed. After the Bmbacmid (polh+) DNA and helper plamid were transformed into DH10βby turns, a recombinant polh+BmNPV carrying foreign gene can be rapidly constructed using Bac-to-Bac system. [3] Expression efficiency was increased through knockout of a main proteinase gene coded by BmNPV. The cyctein proteinase (CP) gene was knocked out of the HyNPV (a host-expanded BmNPV) through homologous recombinatoin.Further, besides the BmNPV Bac-to-Bac system, HyNPV Bac-to-Bac expression system was also successfully established. The Hybacmid was constructed through replacement of polh gene by the above mentioned Kan-miniF-(attTn7)lacZ fragment.This study has big scientific significance. It lies in (1) It offered a novel method to generate recombinant BmNPV by site-specific transposon in E.coli, challenging the traditional idea that recombinant BmNPV virus must be generated in insect larva or cultured insect cells. The BmNPV Bac-to-Bac system avoided the tedious, time-consuming plaque assay, so this new system was more efficient and shows good prospect in application. It provides a novel technical platform. (2) The BmNPV Bac-to-Bac (polh+) expression system can realize the oral infection. (3) The foreign protein production could be highly increased through reduction of protein degradation caused by proteinase.