Preparation method of recombinant porcine circovirus type 2 Cap antigen

A porcine circovirus and antigen technology, applied in the directions of virus antigen components, botanical equipment and methods, microorganism-based methods, etc., can solve the problems of high production cost and unverified immune effect, and achieve low production cost and good clinical effect. , to avoid lost effects

Inactive Publication Date: 2011-07-20
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The methods used by some domestic universities and scientific research institutions to prepare porcine circovirus type 2 subunit vaccines include constructing recombinan...

Method used

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  • Preparation method of recombinant porcine circovirus type 2 Cap antigen
  • Preparation method of recombinant porcine circovirus type 2 Cap antigen
  • Preparation method of recombinant porcine circovirus type 2 Cap antigen

Examples

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Embodiment 1

[0028] The plasmid pGAPZαA is used as the yeast expression vector, and yeast X33 is used as the expression host bacterium. The preparation method includes the following steps:

[0029] 1. The virus was isolated from the lungs of pigs infected with circovirus type 2, and the whole genome was amplified to obtain the target gene ORF2. The upstream primer is SEQ ID NO: 1, the downstream primer is SEQ ID NO: 2, and the reaction conditions are: 94°C pre-denaturation for 5 minutes, then start 30 cycles, 90°C for 30S, 50°C for 40S, 72°C for 45S, and finally 72°C for extension 10min.

[0030] 2. Ligation and transformation of PCV2 ORF2 gene and pMD18-T cloning vector. Gently mix 0.5 μL of pMD18-T vector with 4.5 μL of the target gene ORF2 amplified in step 1 and 5.0 μL of SolutionⅠ, and connect overnight at 16°C , and the ligation product was labeled pMD18-ORF2. 5 μL of the ligation product was transformed into 150 μL of DH5α competent cells prepared, and the transformed bacterial so...

Embodiment 2

[0036] The plasmid pGAPZαA is used as the yeast expression vector, and the yeast SMD1168 is used as the expression host bacterium. The preparation method includes the following steps:

[0037] 1. The virus was isolated from the lungs of pigs infected with circovirus type 2, and the whole genome was amplified to obtain the target gene ORF2. The upstream primer is SEQ ID NO: 1, the downstream primer is SEQ ID NO: 2, and the reaction conditions are: 94°C pre-denaturation for 5 minutes, then start 30 cycles, 90°C for 30S, 50°C for 40S, 72°C for 45S, and finally 72°C for extension 10min. The ORF2 gene was modified according to the codon preference of yeast, and the target gene was obtained by whole gene synthesis of Sangon Bioengineering (Shanghai) Co., Ltd. The recombinant plasmid is marked as pBluescript II SK + -ORF2. The target gene sequence is shown in SEQ ID NO: 3, with a total of 734bp.

[0038] 2. pBluescript II SK + -150 μL of DH5α competent cells prepared by transfor...

Embodiment 3

[0046] Example 3. Preparation of subunit vaccines

[0047] Mix one or several ingredients such as preservatives, protective solutions, stabilizers, antibacterials, etc. with the isolated and purified Cap protein, add adjuvants and mix with normal saline or PBS, and adjust the pH of the mixture to the physiological value, that is, pH7.0 ~7.5, constitute the porcine circovirus subunit vaccine. Adjuvants can be selected from aluminum gel, carbopol 971, saponin, liposome, CpG-ODN, nano adjuvant, oil emulsion adjuvant, etc., and can choose to add cytokines such as interferon, interleukin, etc., wherein Cap The active ingredient concentration of the protein should reach 200-350μg / mL, 200-350μg per dose.

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Abstract

The invention discloses a preparation method of a recombinant porcine circovirus type 2 Cap antigen, which comprises the following steps: using a yeast expression system to amplify the PCV2-ORF2 gene; then, constructing an expression engineering bacterium: inserting an amplification sequence into a yeast expression vector to form a recombinant expression vector, linearizing, and transforming into a pichia pastoris host cell so as to finish the construction of the expression engineering bacterium; and finally, carrying out secretory expression on the recombinant microzyme to obtain the recombinant porcine circovirus type 2 Cap antigen protein. The yeast expression system can carry out processing, folding and posttranslational modification on the expressed protein, so that the expressed protein has biological activity. Compared with Escherichia coli, the yeast expression system has more advantages; and compared with a mammal cell expression system, the yeast expression system is more convenient to operate. Compared with Saccharomyces cerevisiae, the pichia pastoris can not be easily subjected to hyperglycosylation, thereby facilitating the separation and purification steps. By using the yeast expression system, the invention has the advantages of simple operation process, high expression quantity and low production cost, can implement large-scale production, and is beneficial to the popularization and application of the porcine circovirus subunit vaccine.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing recombinant porcine circovirus type 2 Cap antigen and a vaccine prepared based on the Cap antigen expressed by yeast. Background technique [0002] PCV2 is one of the smallest viruses newly found to infect pigs in recent years. Clinically, it mainly manifests as emaciation, pale skin, slow growth, dyspnea, dermatitis, diarrhea, jaundice, etc. Since the first report in Canada in the 1990s, the disease has It is widely popular in the world, and has caused huge losses to the pig industry in various countries in the world, and my country, a big pig-raising country, has also suffered greatly from it. Studies have shown that porcine circovirus disease and many factors work together to cause the disease. When the disease invades the body together with other pathogens, it is conducive to the replication and proliferation of PCV2 in the target cells, thereby causing imm...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/33C12P21/02A61K39/12A61P31/12C12R1/84
Inventor 黄毓茂刘娜吴锋刘德辉
Owner SOUTH CHINA AGRI UNIV
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