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Animal genetic engineering interferon alpha and gamma composite preparations and production method and clinical application thereof

A technology of interferon α and genetic engineering, applied in genetic engineering, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of product loss, denaturation and renaturation, and increased cost of enzyme digestion, etc., to increase expression , strong antiviral activity, and strong immunomodulatory activity

Inactive Publication Date: 2012-02-01
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Denaturation and enzymatic digestion not only increase the cost, but also cause serious product loss

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The production method of chicken genetic engineering interferon α and γ compound preparation is specifically carried out by the following steps:

[0035] A. Extract chicken peripheral blood or splenic lymphocytes, culture in vitro and induce with ConA inducer, extract cell total RNA by Trizol method, design specific primers, clone chicken interferon α and γ genes by RT-PCR technology, and use T-A strategy Ligate it to the pGEM-T vector;

[0036] B. Redesign the primers, add restriction sites at the 5′ ends of the upstream and downstream primers, add BamH I and HindIII to chicken interferon α, add Eco I and Sph I to chicken interferon γ, and use T vector as a template to PCR amplify chicken interferon The expression fragments of α and γ genes were respectively connected to the vector pFastBac by double enzyme digestion TM On Dual, the gene expression fragment encoding chicken interferon α is placed under the control of the promoter Polyhedrin promoter, the gene express...

Embodiment 2

[0043] The production method of the compound preparation of porcine genetically engineered interferon α and γ specifically consists of the following steps:

[0044] A. Extract porcine peripheral blood or splenic lymphocytes, culture in vitro and induce with ConA inducer, extract total RNA from cells by Trizol method, design specific primers, clone porcine interferon α and γ genes by RT-PCR technology, and use T-A strategy Ligate it to the pGEM-T vector;

[0045] B. Redesign the primers, add restriction sites to the 5′ ends of the upstream and downstream primers, add EcoR I and HindIII to porcine interferon α, add Nco I and Sph I to porcine interferon γ, and use T vector as a template to PCR amplify porcine interferon The expression fragments of α and γ genes were respectively connected to the vector pFastBac by double enzyme digestion TM On Dual, the gene expression fragment encoding porcine interferon α is placed under the control of the promoter Polyhedrin promoter, the ge...

Embodiment 3

[0052] The production method of the compound preparation of bovine genetically engineered interferon α and γ specifically consists of the following steps:

[0053] A, extract bovine peripheral blood lymphocytes, through in vitro culture and induction of ConA inducer, Trizol method extracts total RNA of cells, designs specific primers, clones bovine interferon α and γ genes by RT-PCR technology, and cloning them by T-A strategy Ligated to pGEM-T vector;

[0054] B. Redesign the primers, add restriction sites to the 5′ ends of the upstream and downstream primers, add BamH I and HindIII to bovine interferon α, add Nco I and Sph I to bovine interferon γ, and use T vector as a template to PCR amplify bovine interferon The expression fragments of α and γ genes were respectively connected to the vector pFastBac by double enzyme digestion TM On Dual, the gene expression fragment encoding bovine interferon α is placed under the control of the promoter Polyhedrin promoter, the gene ex...

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PUM

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Abstract

The invention relates to an animal genetic engineering interferon compound preparation, the production method and the clinical application. The animal genetic engineering interferon compound preparation is prepared by extracting the animal peripheral blood or spleen lymphocytes, culturing, inducing by in vitro inductor, extracting the cell total RNA by Trizol, designing the specific primer, cloning the interferon gene by RT-PCR technology and connecting to the pGEM-T carrier by T-A strategy; the primer is designed again, the both ends are added with different restriction endonucleases digestion sequences, initiation codon ATG and termination codon TAA, leading peptide sequences are removed, the PCR amplification is carried out by taking the recombination T carrier as the template, so as to obtain the expression fragment of the animal interferon gene; the expression fragment is carried out with the rare codon mutation, and gel recovery target fragment is connected on the carrier of pFastBacDual with the same double restriction endonucleases digestion after double restriction endonucleases digestion; the interferon-Alpha of an animal is cloned to multiple cloning sites under the control of Polyhedrin promoter, the interferon-Gamma of the same species animal is cloned to multiple cloning sites under the control of p10 promoter; the constructed carrier of pFastBacDual plus interferon Alpha plus interferon Gamma are transfected to DH10BacE.coli to carry out recombination; the constructed recombinant bacmid is extracted and purified after the blue white screening and the PCR identification, and the transfection of SF9 insect cell is carried out by a liposome method; the expression product is carried out with identification and purification; the animal genetic engineering interferon compound preparation is carried out with anti-virus activity detection and the evaluation of the clinical application effects.

Description

technical field [0001] The present invention relates to a compound preparation of animal genetic engineering interferon and its production method and application. The compound preparation of animal genetic engineering interferon is controlled by different promoters on the same carrier with animal interferon α and γ genes respectively. Interferon complex product obtained by independent expression in host cells. Background technique [0002] The outbreak of a series of livestock and poultry viral infectious diseases such as avian influenza and foot-and-mouth disease has caused huge losses to the global society and economy, directly threatening food safety and human health. In order to control and eliminate these animal diseases, the conventional way is to prevent and use antibiotics through vaccine immunization, but due to the poor breeding environment, the virus is constantly mutating. The use of antiviral chemical drugs and the strengthening of drug residue detection in foo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/21C12N15/86C12N15/12A61P31/14A61P31/20C12Q1/68
CPCY02A50/30
Inventor 王彦彬崔保安夏平安王亚宾张红英
Owner HENAN AGRICULTURAL UNIVERSITY
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