Engineered bombyx mori baculovirus vector and method of increasing the NS1 expression amount by utilization of the vector

Abstract

The invention discloses an engineered bombyx mori baculovirus vector. The vector is obtained by replacing chitinase and cysteine protease genes in a bombyx mori baculovirus vector Bm-Bacmid with a Cm expression cassette and an egfp expression cassette which are in series connection. The egfp gene in the engineered vector is subjected to integrated expression, and can be used for monitoring cell transfection or infection and conditions after insects are infected in real time. By utilization of the vector, a bombyx mori bidensovirus nsl gene expression cassette is inserted into a transposition site, and a BmN cell or bombyx mori is infected by the prepared recombinant virus, so that expression of a targeting gene can be obtained. Results show that: the vector lacks Chitinase and Cystein Protease genes, delays cell lysis and insect liquidation, facilitates expression of an exogenous gene and inhibits digestion of a targeting protein to a certain degree, thus increasing the expression amount of the nsl gene and laying sound foundations for deep research of the structure and functions of the NS1 protein.

Description

technical field [0001] The invention relates to genetic engineering technology, and discloses a construction process of a novel silkworm baculovirus expression vector and its application in protein expression. Background technique [0002] Baculovirus-insect cell expression system (BEVS) prepares recombinant baculovirus carrying foreign genes, infects insects or insect cells to express foreign proteins, and the expression products have correct spatial folding and glycosylation modifications, Its biological activity is close to that of its corresponding natural products. In BEVS, A. californica nuclear polyhedrosis virus (AcNPV) or silkworm nuclear polyhedrosis virus (BmNPV) are commonly used as vectors for recombinant virus construction. When expressing foreign proteins, we tend to choose silkworm baculovirus - Insect cell expression system, which is closely related to the rich silkworm resources and large-scale breeding in my country (Maeda et al., Nature, 1985, 315:592–594...

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Problems solved by technology

[0004]Despite the many advantages of the BEVS system and the continuous improvement in technology, it still faces some challenges in practice, such as expressing foreign proteins in cultured insect cells, Its production cost is relatively expensive (Tiwari et al., Mol Biotechnol, 2010, 46:80–89); although the cost of expressing exogenous proteins in insects such as silkworms is relatively low, it increases the cost of isolation from insects and target protein The difficulty of purification; the lack of effective fluorescent signals in the BEVS system makes it difficult to quickly determine whether infectious recombinant virus particles are produced in the transfected cells, which affects the process of foreign protein expression; in addition, compared with the prokaryotic expression system, BEVS The expressed exogenous protein has a relatively low yield, especially after the infected cells will eventually be lysed, so that the expression level of the target protein and its modification have not yet reached the optimal state (Ikonomou et al., Appl Microbiol Biotechnol, 2003, 62 :1–20), while replacing the polyhedrin late promoter with the ie1 early promoter can continuously express the target protein, and its glycosylation modification can be efficiently processed, but the expression level of the target protein is reduced (Jarvis et al., Biotechnology ,1990,8:950–955), these shortcomings limit the use of BEVS system to some extent

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