Soluble human program death protein-1-lgV and preparation method thereof

A programmed death and soluble technology, applied in the field of medicine and biology, can solve problems such as exacerbation of diseases

Inactive Publication Date: 2008-07-09
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unlike NOD mice, blocking PD-L2 but not PD-L1 led to disease exacerbation

Method used

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  • Soluble human program death protein-1-lgV and preparation method thereof
  • Soluble human program death protein-1-lgV and preparation method thereof
  • Soluble human program death protein-1-lgV and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0073] 4. Construction, expression and purification of fusion protein PD-1-IgV

[0074] 4.1 Design of PD-1-IgV gene

[0075] The structure of PD-1 in the Swiss-Pro protein database was analyzed, combined with the literature, the amino acid sequence of the PD-1 extracellular fragmented IgV domain was intercepted, and the amino acid sequence of the PD-1 extracellular fragmented IgV (named: hPD-1-IgV) was obtained. According to the human PD-1-IgV gene sequence published by Gene-bank, the 5-end was inserted into the BamHI restriction site, and the 3-end was introduced with a stop codon TGA and inserted into the SalI restriction site. According to the codon preferred by Escherichia coli, the purpose of synthesis was Gene. synthetic gene sequences,

[0076] GGA TCC CAG TAT ATA AAA GCA AAT TCT AAA TTT ATA GGT ATA ACT GAAGCA GCA GCA GAA TTC GAC CTA TAT GTG GTA GAG TAT GGT AGC AAT ATG ACAATT GAA TGC AAA TTC CCA GTA GAA AAA CAA TTA GAC CTG GCT GCA CTA ATTGTC TAT TGG GAA ATG GAG G...

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Abstract

The invention discloses soluble programmed dead protein-1-IgV and a preparation process. The protein sequence of is showed as followed: PPTFFPAL LVVTEGDNAT FTCSFSNTSE SFVLNWYRMS PSNQTDKLAA FPEDRSQPGQ DSRFRVTQLP NGRDFHMSVV RARRNDSGTY LCGAISLAPK AQIKESLRAE LRVTERRAEV PTAHPSP. The invention obtains gene of Hpd-1-IgV through applying artificial synthesis according to the structure of human PD-1, target gene is cloned into pQE-30 pronucleus expression carrier to transform colibacillus and is expressed in colibacillus with high efficiency, bacterioclasis shows that target protein is existed in inclusion body form, and purified soluble programmed dead protein-1-IgV is finally obtained through inclusion body scouring, affinity chromatography, gel column chromatography and dialysis renaturation. The soluble programmed dead protein-1-IgV can respectively combine with mPD-L1/Fc and hPD-L1/Fc in extra-corporal shPD-1IgV and on cell level and molecular level, and has certain tumor killing effect in internal shPD-1IgV. Therefore, shPD-1IgV has excellent internal and external biological activity, provides experiment basis for curing tumor, establishes experiment foundation, and has potential application prospect.

Description

technical field [0001] The invention belongs to the field of medical and biological technology, and relates to technologies such as gene synthesis, protein expression and purification, in vitro activity detection, and animal tumor inhibition detection, in particular to a soluble human programmed death protein-1-IgV (hPD-1-IgV) and its preparation method. Background technique [0002] The body's immune system can identify "self / non-self", conduct immune surveillance, identify and respond to "non-self", and even eliminate it. However, this powerful immune protection system of the body is helpless in the face of tumors. Tumors can still develop and metastasize under the powerful immune function of the human body, indicating that tumors have their own protective mechanisms. Tumor cells can evade the body's immune recognition and attack by modifying their own surface antigens and changing the microenvironment around the tumor tissue, which is tumor immune escape. There are man...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C12N15/12C12N15/70
Inventor 张英起吴守振王伟华韩苇包春杰薛晓畅李萌秦鑫
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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