Method for constructing BmNPV- silkworm larvae multiple gene expression system

Abstract

The invention relates to a method for constructing a BmNPV-domestic silkworm larva polygene expression system, which comprises the following steps: (1) an I-SceI expression cassette controlled by arabinose promoter (pBAD) is recombined into the genome of E.coli D H10B to obtain new recipient bacteria I-SceI DH10B; (2) a donor plasmid used for combined transformation is constructed; (3) a recombination site of cre / loxp replaces v-cath gene and chitin gene in the BmBacmid genome, and two I-SceI sites replace p70 gene in the BmBacmid to obtain a receptor I-sceI BmMultibac; (4) an exogenous gene is induced by the three methods of cre-loxp recombination, Tn7 recombination and combined transformation; (5) a recombined Bacmid DNA is prepared by a conventional method, and is used for transfecting insect cells or injected into the silkworm larva. The BmNPV-domestic silkworm larva polygene expression system lays a foundation for studying multimeric proteins, the interaction between proteins and preparing virus-like particles.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and protein engineering, and in particular relates to a method for constructing a BmNPV-bombyx mori larvae multigene expression system. Background technique [0002] Because it is safe for humans and mammals, the super-strong polyhedron promoter (polh) drives the high-efficiency expression of foreign genes in insect cells, the insect cell culture is convenient, the virus operation is simple, and the ability to clone foreign fragments is large (30-50kb) , The expression product has the characteristics of correct post-processing, etc., and the Baculovirus Expression System (BES, Baculovirus Expresson System) has become one of the excellent expression systems for expressing eukaryotic genes. At present, the sources of exogenous genes expressed by the baculovirus expression system are found in various organisms such as viruses, bacteria, fungi, animals and plants, and its application scope involves...

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Problems solved by technology

[0003] However, the BmNPV-bombyx mori larvae expression system also has its disadvantages: due to the size limitation of the transfer vector, a donor plasmid can only carry four exogenous gene expression cassettes at most, which is useful for the simultaneous expression of multiple exogenous genes using the BmNPV-bombyx mori larvae expression system. great limitations

Another method is to simultaneously clone multiple target genes into the NPV genome through a donor plasmid carrying multiple promoters. Only one recombinant baculovirus is needed to infect insect cells to express several target proteins, and then assemble Formation of VLPs. In the early days, the previous method was the main method. This method is cumbersome to operate and requires the construction of several recombinant baculoviruses. During the infection process, it is difficult to ensure that multiple baculoviruses enter each cell at the same time, which may cause VLPs assembly. less amount

With the development of recombinant baculovirus technology, especially the emergence of multiple (2-4) expression cassettes at the same time, the latter technology is currently used to obtain VLPs. The operation is relatively simple and the output of VLPs is high, but Due to the limitations of the BmNPV-bombyx mori larvae expression system in the expression of multiple genes, the widespread utilization of its production of VLPs is limited

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