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Establishment of protein secreted expression vector and application of same

An expression vector and secreted technology, applied in the field of genetic engineering, can solve the problems of no purification tag, unfavorable recombinant protein purification, high protein content, etc.

Inactive Publication Date: 2012-05-16
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sf9 or Sf21 are often used to prepare recombinant viruses because of their high transfection efficiency, and can also be used to amplify recombinant proteins; while High 5 cells have low transfection efficiency, but can be used to amplify recombinant proteins in large quantities using spinner bottles due to their suspension culture characteristics
[0009] At present, the use of baculovirus white expression system to express foreign proteins is basically intracellular expression. The expression vector does not contain protein signal peptide secretion signals, nor does it contain purification tags, so that the expressed recombinant protein cannot be secreted outside the cell. The high protein content and complex components bring difficulties to protein purification and increase the cost, which is not conducive to the purification of recombinant proteins

Method used

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  • Establishment of protein secreted expression vector and application of same
  • Establishment of protein secreted expression vector and application of same
  • Establishment of protein secreted expression vector and application of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Construction of Secreted Baculovirus Donor Plasmid

[0033] Firstly, the gp67 signal peptide sequence was designed and synthesized, and 6×His and 3×flag tags were added to facilitate protein identification (synthesized by Shanghai Sangon Bioengineering Co., Ltd.). Add a PreScissionTM protease cleavage site between the 6×His and 3×Flag tags, and use the cleavage activity of the PreScission protease after expressing the fusion protein to cut the His and Flag tags from the target protein. The length of the synthesized sequence was 595bp, and it was inserted into the pUC57 vector. The insertion sites were BssHII and MfeI, and a recombinant plasmid containing the gp67 signal peptide sequence was prepared, which was named pUC57-gp67.

[0034] The baculovirus expression vector pFastBac 1 (Invitrogen, USA) was double-digested with BssHII and MfeI (New England Biolabs). The system was: 5 μl of pFastBac 1, 1.5 μl of BssHII and MfeI, 5 μl of 10× buffer, H 2 O 37 μl, in...

Embodiment 2

[0037] Example 2. Construction of secreted baculovirus expression vector containing influenza A virus HA1 gene

[0038] The secreted baculovirus donor plasmid pFBgp67 prepared above was digested with Xho I and Hind III (New England Biolabs). The system was: 5 μl of pFBgp67, 1.5 μl of Xho I and Hind III, 5 μl of 10× buffer, h 2 O 37 μl, incubated at 37°C for 2 hours, and the carrier fragment was recovered with Qiagen's agarose gel recovery kit.

[0039] At the same time, Xho I and Hind III were used to double-digest the HA1 gene subtype containing 16 influenza virus genotypes (according to the corresponding gene sequence of GenBank, entrust Shanghai Sangong Bioengineering Company to synthesize, the influenza virus is divided into 16 subtypes according to the HA gene, HA1 The gene must be cut into HA1 and HA2 to be functional. These 16 HA1 genes are the HA1 genes of 16 subtypes of influenza virus. Their sequences have certain similarities, and the homology is between 28-77%). ...

Embodiment 3

[0041] Example 3. Transposition reaction of secreted recombinant baculovirus expression vector

[0042] Take out DH10Bac competent cells (Invitrogen, USA) from the -80°C refrigerator and put them on ice to melt, add 1 μl of recombinant expression vector plasmids pFBgp67-H1HA1 to pFBgp67-H16HA1, flick the eppendorf tube to mix well, and place in ice bath for 30 minutes Afterwards, heat shock at 42°C for 90 seconds, then place in an ice bath for 5 minutes, add 900 μl of SOC culture solution, place at 37°C and shake at 200 rpm for 4-6 hours, take 40 μl of bacterial liquid to coat Luria Agar (Invitrogen, USA) selective culture plate (kanamycin 50 μg / ml, gentamycin 7 μg / ml, tetracycline 10 μg / ml), cultured at 37°C in the dark for at least 48 hours, and observed the growth of blue and white spots.

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Abstract

The invention provides a baculovirus expression vector capable of realizing secreted expression of protein and a method for establishing the same, as well as a method for producing protein by using the vector.

Description

technical field [0001] The invention relates to the field of genetic engineering. Specifically, the present invention relates to a baculovirus expression vector capable of secreting and expressing a protein, a construction method thereof, and a method for producing a protein using the vector. Background technique [0002] Exogenous protein expression systems include prokaryotic cell systems and eukaryotic cell systems. The prokaryotic cell system is mainly Escherichia coli cells. It is easy to operate and the expression product is stable, but it cannot perform some post-translational processing and modification on the expressed protein. Eukaryotic cell systems include mammalian cells, yeast cells, and insect cells, among others. Insect cell expression system (ie, baculovirus expression system) is an expression system that uses insect baculovirus as a foreign gene carrier and insect cells as a receptor. Compared with bacterial, yeast and mammalian cell expression systems, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/866C12N15/64C12N7/01C12P21/00
Inventor 王健伟崔淑娟郭丽
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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