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Porcine alpha interferon and interleukin 2 chimeric gene, construction method and protein purification method thereof

A technology of interleukin and alpha interferon, which is applied in the field of flexible gene connectors to achieve the effect of good prevention and control and simplified operation process

Inactive Publication Date: 2009-11-04
HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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Problems solved by technology

Studies have shown that the prokaryotic expression of genetically engineered recombinant porcine interferon alpha (rPoIFN-α) protein and recombinant porcine interleukin-2 (rPoIL-2) protein has antiviral activity, and rPoIL-2 also has the ability to enhance the body's cellular immunity so far As a result, there are no similar or identical reports on the construction, fusion expression and purification of porcine interferon-alpha and interleukin-2 chimeric genes at home and abroad, and there is no commercial genetically engineered recombinant porcine interferon-alpha / interleukin-2 gene. PoIL-2 fusion protein (rPoIFN-α-linker-PoIL-2) can be applied

Method used

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  • Porcine alpha interferon and interleukin 2 chimeric gene, construction method and protein purification method thereof
  • Porcine alpha interferon and interleukin 2 chimeric gene, construction method and protein purification method thereof
  • Porcine alpha interferon and interleukin 2 chimeric gene, construction method and protein purification method thereof

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Embodiment Construction

[0040] 1 method

[0041] 1.1 Design and synthesis of primers

[0042] The gene linker is a flexible linker rich in glycine (G) and serine (S) (G 4 S) 3 , a total of 15 amino acids. According to the sequence of the PoIFN-α gene in rpQE-30 / PoIFN-α (GenBank accession number: AB369102) and the sequence of the PoIL-2 gene in GenBank (AB194099), we designed 4 primers for SOE using Oligo6.0 software -PCR amplification, the sequence is as follows: P1:

[0043] 5′-TCA GCATGC TGTGACCTGCCTCAGACCC-3' (with SphI restriction site); P2:

[0044] 5′-

[0045] GCCACCGCCAGAGCCACCTCCGCCTGAACCGCTCCACCCTCCTTCTTCCTGAGTCTG-3′, the full length is 58bp, of which the partial linker length is 39bp, which is the downstream primer used to amplify the PoIFN-α-linker part of the PoIFN-α-linker-PoIL-2 chimeric gene; P3: 5′-GGCGGTTCAGGCCTGGAGGTGGCTCTGGCGGTGGCGGATCGGCACACCTACTTCA ', the upstream primer used to amplify the linker+PoIL2 part of the chimeric gene; P4: 5'-TAC GGATCC AGTCAGTGTTGAGTAGATGC-...

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Abstract

The invention belongs to the technical field of biological genetic engineering, and discloses technology for constructing and expressing porcine alpha interferon (PoIFN-alpha) and interleukin 2 (PoIL-2) chimeric gene and quickly renaturing and purifying expression protein. The technology constructs mature peptide genes of PoIFN-alpha and PoIL-2 into PoIFN-alpha-linker-PoIL-2 chimeric gene through a genetic flexible linker (linker) (G4S)3 and clones the chimeric gene into a pGEM-T Easy vector by adopting an overlap extension PCR method, and subclones the chimeric gene into a pQE-30 expression vector for prokaryotic expression. The recombinant fusion protein (rPoIFN-alpha-linker-PoIL-2) can be quickly renatured and purified through urea modification, renaturation by low concentration protein renaturing solution, PBS solution dialysis and other steps. The purified rPoIFN-alpha-linker-PoIL-2 fusion protein has proliferation activity for inhibiting vesicular stomatitis virus (VSV) on a cell, is used for prevention and treatment of porcine virosis as a main ingredient of an antivirus preparation, and has high efficiency, broad spectrum, safety and low price.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and relates to the construction of chimeric genes of porcine alpha interferon and interleukin 2, fusion expression and purification technology, and also relates to a flexible gene linker. Background technique [0002] my country is a big pig raising country in the world. There are many kinds of pig diseases, especially the highest morbidity and mortality caused by viral infectious diseases, which cause huge economic losses to the pig industry every year. The viruses that are more harmful to the pig industry mainly include: swine fever, porcine reproductive and respiratory syndrome, porcine circovirus type II, swine influenza, porcine parvovirus, porcine foot-and-mouth disease, porcine pseudorabies, porcine transmissible gastroenteritis, Porcine epidemic diarrhea and other viruses. Among them, the first five kinds of viruses are the main source of disease causing respiratory syndro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/11C12N15/70C07K19/00C07K1/14A61P31/14C12R1/19
Inventor 吴志明张志凌张健闫若潜刘光辉盛敏苗连叶方先珍谢彩华赵明军赵雪丽程俊贞刘梅芬
Owner HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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