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Genetic engineering subunit vaccine of goat contagious pleuropneumonia as well as preparation method and application thereof

A gene and goat technology, applied in the field of animal immune medicine, can solve the problems of limited popularization and application, many types of mycoplasma clusters, and the quality of antigens is greatly affected by culture conditions, and achieves good cross-protection, reduced production costs, and high expression levels. Effect

Inactive Publication Date: 2021-10-08
SUZHOU MIDI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, my country mainly relies on inactivated vaccines to prevent CCPP. However, the original vaccine uses strong poisonous tracheal injection of goats, and the lung tissue is collected to make a vaccine. Allergic reactions to vaccines
At the same time, due to the many types of mycoplasma clusters to which the disease belongs, the cross-protection between strains is not ideal, and the quality of the antigen is greatly affected by the culture conditions, which limits its popularization and application.

Method used

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  • Genetic engineering subunit vaccine of goat contagious pleuropneumonia as well as preparation method and application thereof
  • Genetic engineering subunit vaccine of goat contagious pleuropneumonia as well as preparation method and application thereof
  • Genetic engineering subunit vaccine of goat contagious pleuropneumonia as well as preparation method and application thereof

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preparation example Construction

[0062] For example, in a specific embodiment of the embodiments of the present invention, a method for preparing a genetically engineered subunit vaccine for goat infectious pleuropneumonia may specifically include:

[0063] (1) preparing nucleic acid molecules for encoding the aforementioned first recombinant protein (recombinant GK protein) and the aforementioned second recombinant protein (recombinant MagT protein);

[0064] (2) respectively cloning the nucleic acid molecules encoding the first and second recombinant proteins prepared in step (1) into a shuttle vector to obtain a recombinant shuttle vector containing the gene of interest;

[0065] (3) transform the recombinant shuttle vector obtained in step (2) into DH10Bac bacteria, select the recombinant bacteria, extract the genome and transfect Sf9 cells (or other aforementioned insect cells), to obtain recombinant baculovirus;

[0066] (4) cultivating the Sf9 cells (or other aforementioned insect cells) and then recom...

Embodiment 1

[0072] Example 1 Construction and Identification of Transfer Vector pF-GK

[0073] 1. GK gene amplification and purification

[0074] The codon-optimized GK gene (SEQ ID NO: 1) was synthesized in Nanjing GenScript Biotechnology Co., Ltd. and cloned into the pUC17 vector to obtain the pUC-GK plasmid vector. The pUC-GK plasmid was used as a template, and GK-F and GK-R were used as upstream and downstream primers for PCR amplification (the gene sequences of GK-F and GK-R are shown in SEQ ID NO: 3 and 4). For the amplification system, see Table 1.

[0075] Table 1 GK gene amplification system

[0076]

[0077] The reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 45 seconds, 54°C annealing for 45 seconds, 72°C extension for 1 minute, 35 cycles; 72°C extension for 10 minutes.

[0078] Perform gel electrophoresis on the PCR product to verify the size of the target gene, such as figure 1 As shown, the target band appeared at the position of 1...

Embodiment 2

[0096] Example 2 Construction of recombinant baculovirus genome Bac-GK and Bac-MagT

[0097] 1. Transformation of DH10Bac bacteria

[0098] Take 1 μl of the pF-GK plasmid and 1 μl of the pF-MagT plasmid in Example 1 and add them to 100 μl of DH10Bac competent cells and mix them evenly. Place them in an ice bath for 30 minutes, heat shock in a water bath at 42°C for 90 seconds, and then ice bath for 2 minutes. Add 900 μl of LB liquid medium containing Amp, cultured at 37°C for 5 hours. After 100 μl of bacterial solution was diluted 81 times, 100 μl of diluted bacterial solution was spread on LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and cultured at 37°C for 48 hours.

[0099] 2. Picking Single Clones

[0100] Use an inoculation needle to pick large white colonies, then streak on LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal, and IPTG, culture at 37°C for 48 hours, and then pick single colonies Inoculate the LB liq...

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Abstract

The invention discloses a genetic engineering subunit vaccine of goat contagious pleuropneumonia as well as a preparation method and application thereof. The vaccine comprises a first recombinant protein, a second recombinant protein and a pharmaceutically acceptable carrier, wherein the first recombinant protein and the second recombinant protein respectively have sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 6. The vaccine provided by the invention is free of any toxicity, high in safety and good in immunogenicity, and can generate relatively strong humoral immunity in a sheep body, antigens expressed by the first recombinant protein and the second recombinant protein have a very good cross protection effect, which can simultaneously resist attacks of mycoplasma capricolum subsp.capripneumoniae, mycoplasma capricomlusubsp.capricolum and mycoplasma ovipneumoniae, the immunized sheep can resist virulent virus attack, and the vaccine can be prepared through large-scale serum-free suspension culture by using a bioreactor, and has advantages of easy quality control, batch-to-batch stability, low production cost and the like.

Description

technical field [0001] The invention relates to a genetic engineering vaccine, in particular to a goat infectious pleuropneumonia genetic engineering subunit vaccine, its preparation method and application, and belongs to the technical field of animal immune drugs. Background technique [0002] Contagious Caprine Pleuropneumonia (CCPP) is a severe respiratory disease caused by Mycoplasma capricolum subsp.capripneumoniae (Mccp), which mainly harms goats and wild sheep. It is most common during sudden changes, and the main clinical symptoms are high fever, cough, progressive emaciation, serous and fibrinous inflammation of the lung parenchyma, interlobular interstitium and pleura, and obvious hepatic changes in the lungs. CCPP spreads quickly, has high morbidity and mortality, and causes large economic losses and potential threats to the goat industry in many regions. It is listed as one of the legally notifiable animal diseases by the World Organization for Animal Health (OIE...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C07K14/30C12N15/54C12N15/31C12N15/866A61K39/02A61P31/04G01N33/569G01N33/573
CPCC12N9/1205C07K14/30C12N15/86A61K39/0241A61P31/04C12Y207/0103G01N33/56933G01N33/573C12N2710/14143C12N2800/105A61K2039/552G01N2333/91215
Inventor 方鹏飞金佳颖曹文龙滕小锘张大鹤孔迪
Owner SUZHOU MIDI BIOTECH CO LTD
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