Efficient fusion expression carrier

A technology that integrates expression and vectors, applied in the field of bioengineering, can solve problems such as loss, research and production difficulties, and achieve the effect of simplifying purification work and reducing mutual interference

Inactive Publication Date: 2007-12-26
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing expression vectors are divided into non-fusion expression vectors, fusion expression vectors, co-expression vectors, etc. Vectors can be selected according to different experimental designs. However, there are still many foreign genes that cannot be efficiently expressed by existing expression vectors For the target protein, it is necessary to try a variety of different expression vectors, resulting in unnecessary losses and difficulties for subsequent research and production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of fusion expression vector pEspA

[0031] 1. Amplification of EspA gene

[0032]1) Design and synthesis of primers (restriction sites are underlined)

[0033] According to the espA gene nucleotide sequence (AE005174) of the international standard strain EHEC O157:H7 EDL933 published by GenBank, a pair of primers were designed according to the restriction site search and primer design principles, the upstream primer (SEQ ID NO: 9) 5' The NcoI restriction site was introduced into the end, and the Linker (YAPQDP) sequence (SEQ ID NO: 2) was counted at the 5' end of the downstream primer (SEQ ID NO: 10), and a BamH I site was introduced, and the sticky end digested with BamH I The correct connection between the two sequences can be carried out.

[0034] Upstream primer P1 (SEQ ID NO: 9):

[0035] 5' CCATGG ATACATCAAATGCAAC-3'(NcoI)

[0036] Downstream primer P2 (SEQ ID NO: 10):

[0037] 5'- GGATCC TGCGGCGCGTATTTACCAAGGGATATT-3' (BamHI)

[00...

Embodiment 2

[0065] Example 2 Fusion expression vector pEspA expresses fusion protein EspA-Stx2B

[0066] 1. stx2B gene PCR amplification:

[0067] 1) Obtain the EHEC O157:H7 stx2B gene through GenBank, use the Primer Premier 5.0 software to design the primer sequence, the upstream primer (SEQ ID NO: 13):

[0068] 5′- GAATTC AAAGAAGATGTTTATGGCGGT-3', an EcoR I restriction site was added to the 5' end, and the downstream primer (SEQ ID NO: 14): 5'- CTCGAG GTCATTATTAAACTGCACTT-C-3′, Xho I restriction site was added to the 5′ end, and the primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

[0069] 2) Take 10ml of EHEC O157:H7 cultured overnight, centrifuge to remove the supernatant, add 2ml of autoclaved double distilled water to suspend, boil for 10 minutes, centrifuge at 12000g to take the supernatant as a PCR template. Take 1 μl as a template, pre-denature at 94°C for 10 minutes, react in 94°C for 60s, 55°C for 60s, and 72°C for 60s for 30 cycles, and finally extend ...

Embodiment 3

[0077] Example 3 Fusion expression vector pEspA expresses fusion protein EspA-IntiminC300

[0078] 1. IntiminC300 gene PCR amplification:

[0079] 1) According to the IntiminC300 gene of O157:H7 (SaKai standard strain) found from GenBank, use the software Primer Premier5.0 to design and analyze primers. Upstream primer P1 (SEQ ID NO: 15): 5'- GAATTC TACTTCAGCACTTA-3', downstream primer P2 (SEQ ID NO: 16): 5'- CTCGAG TTCTACACAAACCGCATA-3', the 5' end of P1 introduces the EcoR I restriction site, and the 5' of P2 introduces the Xho I restriction site. Primers were synthesized by Shanghai Yingjun Company.

[0080] 2) Take 2 μl of template, 1 μl each of primers P1, P2 or P3, and P4, 4 μl of dNTPs, 5 μl of buffer, 0.25 μl of BxTaq enzyme, and 32.75 μl of double-distilled water, and react on the PCR machine according to the following cycles: pre-denaturation at 94°C for 5 minutes, Perform 30 cycles at 94°C for 30s→55°C for 30s→72°C for 1min, and finally extend at 72°C for 10mi...

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Abstract

the invention discloses a high-effective prokaryotic fused expressive carrier based on EspA as fusing accomponany, which comprises the following parts: T7 strong promoter, coded EspA accompany protein nucleotide sequence and connecting area, wherein the connecting area contains flexible area, 6-group aminoacidemia purifying point, enterokinase cutting point and multi-clone position.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and particularly relates to a high-efficiency fusion expression vector, which contains a nucleotide sequence encoding an EspA partner protein, and can promote prokaryotic expression when expressed in common host bacteria such as Escherichia coli (E.coli). The system efficiently expresses the exogenous target protein, so that the target protein that is not expressed or has a low expression level can be efficiently expressed. Background technique [0002] In the life science research and the production of biopharmaceuticals and biological products, the preparation of recombinant proteins using expression vectors is an important and critical link. Obtaining a sufficient amount of recombinant protein is a necessary condition for subsequent research and production. Constructing an effective expression vector is a basic requirement for expressing a target gene, and it is also an important facto...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/31C12N15/62C07K14/265C07K19/00
Inventor 毛旭虎邹全明刘艳青王庆旭余抒顾江杨琴易勇杨珺张卫军程建平马颖童文德
Owner ARMY MEDICAL UNIV
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