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Chicken IL-18 (Interleukin-18) and Newcastle disease virus HN (Hemagglutinin-neuraminidase) gene recombinant fusion protein and application

A technology of Newcastle disease virus and fusion protein, which is applied in the field of genetic engineering to produce vaccines and immune adjuvants, can solve the problems of unsatisfactory animal protection experiments, and achieve the effects of easy mass production, good protection, and mass production

Inactive Publication Date: 2012-09-19
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, with the rapid development of molecular biology technology in recent years, the development of NDV genetically engineered vaccines has become a hot spot for people to study, but there are still some unsatisfactory aspects of genetically engineered vaccines in inducing the body's immune response. Animal protection experiments The effect is not ideal

Method used

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  • Chicken IL-18 (Interleukin-18) and Newcastle disease virus HN (Hemagglutinin-neuraminidase) gene recombinant fusion protein and application
  • Chicken IL-18 (Interleukin-18) and Newcastle disease virus HN (Hemagglutinin-neuraminidase) gene recombinant fusion protein and application
  • Chicken IL-18 (Interleukin-18) and Newcastle disease virus HN (Hemagglutinin-neuraminidase) gene recombinant fusion protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1c

[0053] Example 1 Obtaining of chIL-18-HN fusion protein coding gene sequence

[0054] 1. Acquisition of poultry IL-18 gene sequence

[0055] According to the nucleotide sequence of poultry IL-18 in GenBank, a pair of specific primers F was designed using DNA Star7.0 and Primer premier6.0 software 1 , F 2 Amplifies IL-18. Primers were synthesized by Dalian Bao Biological Engineering Company. A NotI endonuclease site was introduced at the F15' end of the primer, a BamHI endonuclease site was introduced at the F25' end and a flexible linker was simultaneously introduced. The primer sequences are as follows:

[0056] F1: SEQ ID NO.1;

[0057] P1: 5′- GCGGCCGCG ATGCCTTTTGTAAGGATA-3′

[0058] F2: SEQ ID NO.2;

[0059] P2: 5′-GGT GGATCC GGCGGTTCTGGCGGATTATAGGTTGTGCCTTTCATTATG-3′

[0060] The NDV-I vaccine was diluted 50 times, inoculated into 10-day-old chicken embryos through the allantoic cavity, 0.2mL / piece, and incubated at 37°C. Undead chicken embryos were taken out...

Embodiment 2c

[0072] Example 2 Construction of chIL-18-HN Fusion Gene Recombinant Yeast Expression Vector

[0073]Use Not I and Kpn I endonucleases to perform double enzyme digestion on the PCR product of the above chIL-18-HN fusion gene and the Pichia pastoris expression vector pPICZαA. After the digestion products are also identified by 1% agarose gel electrophoresis, the gel recovery reagent boxes for recovery identification. The digested chIL-18-HN fusion gene and Pichia pastoris expression vector pPICZαA were ligated overnight at 4°C at a molar ratio of 1:3. Take the ligation product and add it to a polypropylene centrifuge tube containing 100 μL of competent DH5α, mix gently and then ice-bath for 30 min. After the polypropylene centrifuge tube was taken out of the ice, it was heat-shocked at 42°C for 90 sec, and then immediately ice-bathed for 2 min. Add 800 μL of LB medium preheated at 37°C, and shake (100-150 rpm) at 37°C for 45 minutes. Take 100 μL of the bacterial liquid and ev...

Embodiment 3

[0074] Example 3 Construction of a genetically engineered strain expressing chIL-18-HN fusion protein:

[0075] The positive recombinant yeast expression vector pPICZαA-chIL-18-HN obtained in Example 2 was linearized with SacI, and 5 μg of the linearized recombinant expression vector pPICZαA-chIL-18-HN was mixed with 80 μL of competent Pichia pastoris X-33 , transferred to a pre-cooled 0.2cm electro-cup, placed on ice for 5min, 1.5kV, 25μF, 200Ω electric shock, immediately added 1mL of pre-cooled 1mol / L sorbitol, took 200μL and spread it on a YPDS plate, cultured at 30℃ until single Colonies appear; refer to Pichia Expression Kit for detailed steps; use PCR method to analyze Pichia transformants, prepare PCR template by boiling-freezing-boiling method, use primers F1 and F4: the reaction system is the same as above, PCR reaction conditions: 94 ° C for 5 min ; 94°C for 45s, 48°C for 45s, 72°C for 45s, 25 cycles; 72°C for 6 minutes; the clone with a size of about 2200bp was iden...

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Abstract

The invention relates to a preparation method and application of a recombinant chicken interleukin-18 (IL-18) and Newcastle disease virus hemagglutinin-neuraminidase (HN) fusion protein (chIL-18-HN fusion protein). The fusion protein is formed by fusing chicken IL-18 (chiIL-18) protein and Newcastle disease virus HN protein through a flexible Linker peptide; a DNA (Deoxyribonucleic Acid) sequence for coding the fusion protein is inserted into an expression vector pPICZalphaA and yeast X-33 is electrically converted to obtain genetic engineering bacteria for efficiently expressing the recombinant chIL-18-HN fusion protein; the recombinant chIL-18-HN fusion protein is prepared through liquid culture and purification; and the recombinant fusion protein can be used as a Newcastle disease novel genetic engineering vaccine and can also be used as a novel immunologic adjuvant for the Newcastle disease conventional vaccine. Animal immunologic tests prove that the chIL-18-HN fusion protein provided by the invention has the advantages of high safety, no toxic or side effect, capability of effectively enhancing cellular immunity and humoral immunity level of organisms and broad application prospect.

Description

technical field [0001] The invention relates to a fusion protein of chicken interleukin-18 (Interleukin-18, IL-18) and Newcastle disease virus HN, a DNA sequence encoding the fusion protein, a carrier and yeast containing the DNA sequence, and also relates to recombinant fusion The protein and its application belong to the technical field of genetic engineering production of vaccines and immune adjuvants in the biotechnology pharmaceutical industry. Background technique [0002] Newcastle disease (Newcastle disease, ND) is listed by the International Office of Epizootics as one of the most harmful A-type infectious diseases to animals. It is a high-contact, acute septic infectious disease characterized by green loose stools, neurological disorders, and mucosal and serosal hemorrhages; it has caused huge economic losses to the poultry industry. Because of its high morbidity and mortality, Newcastle disease is the number one disease threatening the poultry industry. Whether t...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/62C12N15/81C12N1/19A61K39/17A61K39/39A61P31/14C12R1/84
Inventor 张春杰刘一尘李静程相朝吴庭才李银聚王臣张明亮
Owner HENAN UNIV OF SCI & TECH
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