Method of expressing rotavirus virus protein 6 (VP6) protein by using silkworm bioreactor

A technology of silkworm baculovirus and group A rotavirus, applied in biochemical equipment and methods, botanical equipment and methods, and the use of vectors to introduce foreign genetic material, etc., can solve the problem of low yield and low yield of plant expression systems, Long cycle and other issues, to achieve the effect of broad market prospects, simple operation process, and low production cost

Inactive Publication Date: 2013-03-20
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] The gene length is high, the chemical synthesis is difficult, the yield is low, the generated by-products are difficult to remove, and the synthetic route has many steps; in the biosynthesis, the target protein produced by the general prokaryotic expression vector has very low or no biological activity; the plant expression system Generally, the output is low, the cycle is long, it is difficult to put it into large-scale production, and it is even more difficult to meet the needs of the market; the production of drugs by yeast and animal expression systems is extremely expensive and the output is extremely low, and the same problem exists

Method used

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  • Method of expressing rotavirus virus protein 6 (VP6) protein by using silkworm bioreactor
  • Method of expressing rotavirus virus protein 6 (VP6) protein by using silkworm bioreactor
  • Method of expressing rotavirus virus protein 6 (VP6) protein by using silkworm bioreactor

Examples

Experimental program
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Effect test

experiment example 1

[0083] Experimental example 1, the acquisition of group A rotavirus VP6 gene:

[0084] The full-length VP6 gene of group A rotavirus T114 Chinese local strain is 1356 bp, and the coding sequence is 1194 bp long (as shown in SEQ ID NO: 1), encoding a protein with a molecular mass of 44.9 kD consisting of 397 amino acids. The gene was donated by the Capital Institute of Pediatrics. According to the coding sequence of the obtained VP6 gene, design the 5' forward primer P1 (5'-CGC GGATCC ATGGAGGTTCTGTACTC-3', the underline is the BamHI restriction site) and the 3' reverse primer P2 (5'-CCG GAATTC TCACTTAATCAACATGC-3', the underline is the EcoRI restriction site).

[0085] PCR amplification system:

[0086]

[0087] PCR amplification conditions:

[0088]

[0089] Purify the PCR product to obtain the VP6 gene, the target fragment size is about 1.2kb, see figure 2 .

experiment example 2

[0090] Experimental Example 2, Construction and Identification of Transfer Vector Plasmid

[0091] After the PCR amplification product of the VP6 gene is purified, the purified PCR product is obtained, which is double-digested with restriction endonucleases EcoRI and BamHI, and then inserted through the same digestion (that is, with restriction endonucleases EcoRI and BamHI double-enzyme cut) transfer plasmid pBacPAK 8 (purified carrier plasmid pBacPAK 8), and transformed into Escherichia coli (E. coli) TG1 competent cells for positive colony screening, determined the nucleotide sequence of the recombinant expression plasmid, identified the correct plasmid named pBacPAK 8 -RVVP6.

[0092] recombinant plasmid pBacPAK 8 -RVVP6 was digested by PCR and EcoRI / BamHI double enzymes showed that there was an obvious specific band around 12 00 bp, which was consistent with the expected size, while the negative control did not ( image 3 ), so it can be determined that the target gen...

experiment example 3

[0151] Experimental example 3, construction and screening of BmNPV-RVVP6 of recombinant silkworm baculovirus:

[0152] Extraction of Bombyx mori baculovirus Bm-BacPAK 6 DNA, and digested with Bsu36 I to linearize it to obtain linearized silkworm baculovirus Bm-BacPAK 6 DNA; extract highly pure pBacPAK 8 -RVVP6 plasmid and linearized silkworm baculovirus Bm-BacPAK 6 DNA was co-transfected into silkworm BmN cells.

[0153] details as follows:

[0154] Take A centrifuge tube and add the recombinant transfer vector plasmid pBacPAK 8 - RVVP6 1 μg, linearized viral DNA (i.e., linearized Bombyx mori baculovirus Bm-BacPAK 6 DNA) 5 μg, use serum-free TC-100 medium to make up the total volume to 50 μL; B centrifuge tube plus liposome (FuGENE 6 Transfection Reagent purchased from Roche Company,) 6 μL, use serum-free TC-100 medium Make up the total volume to 50 μL; mix centrifuge tubes A and B evenly, and place at room temperature for 15 min; wash the silkworm BmN cells twice wit...

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Abstract

The invention discloses an acquiring method of a recombinant virus Bm nucleopolyhedrovirus (NPV)-rotavirus virus protein 6 (RVVP6). The method comprises the following steps: firstly, choosing group A rotavirus VP6 genes; secondly, constructing a recombinant transfer vector plasmid pBacPAK8-RVVP6; thirdly, introducing the constructed recombinant transfer vector plasmid pBacPAK8-RVVP6 and linearized Bm-BacPAK6 virus DNA into silkworm cells and co-transfecting the silkworm cells in a liposome mediated mode to acquire recombinant virus BmNPV-RVVP6. According to the method of expressing rotavirus VP6 protein by using a silkworm bioreactor, the technology includes that a silkworm BmN, larvae at the age of five and silkworm pupas are transfected by the BmNPV-RVVP6, recombinant protein VP6 is enabled to be expressed in the silkworm. According to the method of expressing rotavirus VP6 protein by using a silkworm bioreactor, raw materials are easy to acquire, and the method is low in production cost and non-toxic, and provides novel methods for studying and producing novel rotavirus vaccines.

Description

technical field [0001] The invention is a method for producing rotavirus VP6 protein in a new way and a method for obtaining the used recombinant virus BmNPV-RVVP6. Background technique [0002] 1. Silkworm bioreactor: [0003] The silkworm bioreactor uses the silkworm BmNPV expression system to produce exogenous proteins, which have high expression levels of exogenous proteins and good natural properties of the products. It does not have the problems of low production efficiency of exogenous protein in mammalian cell culture system and high requirements for equipment conditions, nor does it have defects such as endotoxin of Escherichia coli, glycosylation of expression products and low biological activity. Due to the many potential advantages of this system, it provides a new way for human beings to produce urgently needed protein drugs, genetically engineered vaccines and genetically engineered insecticides, so it has received extensive attention in recent years. [0004...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/66
Inventor 李司张耀洲
Owner ZHEJIANG SCI-TECH UNIV
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