CN111434774B

CN111434774B - Baculovirus expression vector for relieving high titer inhibition

Info

Publication number: CN111434774B
Application number: CN201910025136.0A
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Shaanxi Bacmid Biological Technology Co ltd
Current assignee: Shaanxi Bacmid Biological Technology Co ltd
Priority date: 2019-01-11
Filing date: 2019-01-11
Publication date: 2023-06-23
Anticipated expiration: 2039-01-11
Also published as: CN111434774A

Abstract

The invention discloses a baculovirus expression vector for relieving high titer inhibition. Baculovirus expression systems do not have a significant dose effect, i.e. high viral titers do not increase the yield of exogenous genes. The reason for this is that high titres induce aggregation of the late expression factor LEF-10, which in turn inhibits expression of late genes. The vector reduces the aggregation tendency of LEF-10 by mutating the 21 st leucine site of LEF-10. The expression level of exogenous genes controlled by the late promoter can be remarkably improved by infecting insect cells with high-titer mutant baculovirus. The improvement of the expression yield can obviously reduce the production cost of enterprises. The baculovirus expression vector can be used in the field of biological product industry, in particular subunit vaccine industry.

Description

Baculovirus expression vector for relieving high titer inhibition

Technical Field

The invention belongs to the technical field of recombinant protein expression, and particularly relates to a baculovirus expression vector for relieving high titer inhibition.

Background

Baculoviruses are double-stranded DNA viruses that specifically infect arthropods, and the noctuid california polynuclear polyhedra virus (Autographa californica nucleopolyhedrovirus, acMNPV) is a model species of baculoviruses. Since the first expression of human interferon-beta gene in insect cells by baculovirus such as Smith GE in 1983 (Mol Cell biol. 1983; 3:2156-65), baculovirus expression vector systems have been widely used in scientific research and production due to their low cost, high yield, and various post-translational modification systems.

In the field of protein expression, it is well accepted that gene expression has a dose-response, i.e., there is a certain positive correlation between the copy number of a gene and the expression level of a protein, but baculovirus expression systems are a special case. Researchers have long found that when insect cells are infected, the expression level of the foreign protein is not significantly increased with the increase of the virus titer; at higher titers the protein yield was instead reduced (Biotechnol bioeng. 1994; 44:710-719).

Baculovirus expression systems are transient expression systems and it is desirable to obtain more protein in a single production batch for industrial use, but since high titer viruses do not have the benefit of yield, MOI 3-5 is typically used to infect cells in production, and cannot increase protein yield by increasing MOI.

LEF-10 is an essential late expression factor for baculovirus (Virus Res.2013; 175:45-51, PLoS one.2016; 11:e 0154835), the specific physiological function of which is currently unknown. Previous studies have found that LEF-10 has a strong tendency to aggregate (Virus genes 2004; 29:191-197). In baculovirus-infected insect cells, it was found that high titer viruses induced LEF-10 to form aggregates, which once formed, had little expression of the foreign gene under the control of the late promoter (PLoS one. 2016; 11:e0154835). This finding suggests one such possibility: that is, baculovirus expression systems do not have a gene dose effect because high viral titers induce LEF-10 aggregation, thereby blocking expression of late promoters.

If the inhibition caused by LEF-10 aggregation can be relieved, the yield of the foreign protein can be improved through high-titer virus infection in industrial production, but no research report for reducing the aggregation tendency of LEF-10 is available so far.

Disclosure of Invention

The invention aims to provide a baculovirus vector for relieving high titer inhibition, and aims to solve the problem that a baculovirus expression system mentioned in the background art does not have gene dosage effect.

According to the invention, the baculovirus expression vector with reduced aggregation tendency of LEF-10 is obtained by mutating the 21 st leucine residue of LEF-10. The expression vector can be used for achieving the purpose of improving the yield of the exogenous protein through high MOI.

In the invention, the leucine residue at 21 st position of LEF-10 can be replaced by amino acid residues such as alanine, methionine, serine, cysteine, valine, lysine, tyrosine and the like. Replacement of leucine with these amino acids reduced the aggregation propensity of LEF-10 to varying degrees.

The invention is realized by taking alanine substitution as an example, amplifying rpsL-AMP resistance gene fragments by using primers (SEQ ID NO:1 and SEQ ID NO: 2) with 50bp homology arms on the upstream and downstream of an amino acid coding region of LEF-10 site 21, electrically transforming the obtained PCR product into E.coli containing Bacmid, realizing homologous recombination in the E.coli by inducing expression of RedET recombinase, and obtaining the Bacmid knocked out by ampicillin resistance screening. The artificially synthesized LEF-10 fragment (SEQ ID NO: 3) with the 21-bit alanine mutation sequence is electrically transformed into E.coli containing the LEF-10L 21 knockout Bacmid, the expression of RedET recombinase is induced again, homologous recombination is realized in the E.coli, and the Bacmid with the alanine substituted LEF-10 is obtained through streptomycin resistance reverse screening.

The invention has the advantages that after the 21 st leucine of LEF-10 is replaced, the protein expression level is obviously higher than that of the expression vector before the transformation of LEF-10 under high infection titer, which proves that the invention can indeed provide a baculovirus expression vector with excellent production characteristics and high titer inhibition elimination.

Drawings

FIG. 1 schematically illustrates the mechanism of inhibition release of baculovirus vectors provided by the present invention.

FIG. 2 shows a decrease in the aggregation propensity of LEF-10 following alanine substitution. LEF-10-GFP, LEF-10L 21A, was found in the third day after insect cell infection as a diffuse distribution in the cells, whereas the wild type appeared as punctate aggregates.

FIG. 3 shows green fluorescent protein GFP expressed by using baculovirus vectors provided by the invention. Cells 2 days after virus infection were analyzed by flow cytometry, and the geometric mean was calculated to obtain the total fluorescence intensity. The results show that the GFP yield expressed by the baculovirus vector provided by the invention is not obviously different from that of the wild type at low titer, but is improved by 55% at MOI 8.

FIG. 4 shows the expression level when other amino acids are substituted. Cells 2 days after virus infection were analyzed by flow cytometry, and geometric mean was calculated to obtain GFP total fluorescence intensity.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The principle of application of the invention (alanine substitution for example) is described in detail below with reference to the accompanying drawings.

1. Leu knockout of LEF-10 at 21 st position on Bacmid

The rpsL-AMP fragment was amplified using primers (SEQ ID NO:1 and SEQ ID NO: 2) with 50bp homology arms upstream and downstream of the 21 st site of LEF-10 and the prpsL-AMP plasmid as a template, yielding 1023bp PCR product.

Transferring the obtained PCR product into an escherichia coli strain HS996 with a RedET plasmid and Bacmid by using an electrotransformation method, inducing to generate recombinase through arabinose, and screening the recombined escherichia coli by using an ampicillin resistance plate to obtain positive clone. The LEF-10 leucine coding sequence at position 21 has been replaced by an rpsL-AMP fragment.

2. Insertion of the amino acid of interest at position 21 of LEF-10

The artificially synthesized LEF-10 fragment (SEQ ID NO: 3) with alanine mutation sequence is electrically transformed into the positive escherichia coli in the previous step, the recombinant enzyme is induced by arabinose again, and the recombinant escherichia coli is reversely screened by a streptomycin resistance plate to obtain positive clones. At this point leucine at position 21 of LEF-10 has been replaced by the amino acid of interest, which in this case is alanine.

Bacmid is extracted from escherichia coli, and is named as L21A after sequencing and identification. L21A is subjected to plasmid miniprep and Bsu36I restriction enzyme digestion linearization and then is used for subsequent experiments.

3. Expression of green fluorescent protein GFP

Linearized L21A was co-transfected with pTriEx-GFP plasmid (GFP gene fragment cloned between pTriEx1.1 NcoI/XhoI sites, GFP gene controlled by baculovirus late promoter P10) into Sf9 insect cells and P0 generation recombinant virus was collected 5 days after transfection. The P0 generation virus was amplified to the P1 generation virus, sf9 cells were infected at 0.25-8 MOI, and cells were collected two days after infection. The green fluorescence intensity of the cells was measured by flow cytometry and the geometric mean was calculated (FIG. 3). After comparison, the green fluorescent protein GFP yield was found to be no significant difference from the wild type at low titers, but increased by 55% at MOI 8. This demonstrates that the baculovirus expression vector provided by the invention can actually improve the yield of exogenous genes at high titer.

The alanine substitution at position 21 of LEF-10 described above is merely exemplary of the present invention, and the effect of several other amino acid substitutions is shown in FIG. 4. The present embodiment is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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Claims

1. A baculovirus expression vector for relieving high titer inhibition, characterized in that the leucine residue at position 21 of the late expression factor LEF-10 of the vector is mutated into alanine, methionine, serine, cysteine, valine, lysine, tyrosine residue.