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Capsid protein phage display particle of recombinant II porcine circovirus as well as preparation method and application thereof

A porcine circovirus and phage display technology, applied in the biological field, can solve problems such as restrictions on the promotion and application of such products, cumbersome yeast expression systems, and difficult protein purification, and achieve low purification costs, simple and convenient storage, and clear genetic background. Effect

Inactive Publication Date: 2014-01-29
CHONGQING ACAD OF ANIMAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the domestic porcine circovirus type II (PCV2) inactivated vaccine production is low and the price is high, which limits the promotion and application of this type of product
Although subunit vaccines have been marketed abroad, which can significantly reduce virological symptoms, they are expensive
In addition, there are reports on the expression of ORF2 protein using yeast and baculovirus, but the yeast expression system is cumbersome and has the problem of protein cleavage, while the baculovirus has the disadvantages of high expression cost and difficult protein purification.

Method used

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  • Capsid protein phage display particle of recombinant II porcine circovirus as well as preparation method and application thereof
  • Capsid protein phage display particle of recombinant II porcine circovirus as well as preparation method and application thereof
  • Capsid protein phage display particle of recombinant II porcine circovirus as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Research on Type II Porcine Circovirus Capsid Protein

[0041] The amino acid sequences of PCV2-Cap registered in different periods and with different GenBank numbers were downloaded from GenBank for comparative analysis. It was found that the similarity of the amino acid sequence encoding PCV2-Cap is high, and the amino acid sequence is relatively conservative, which is consistent with the feature that the PCV2-Cap protein is a structural protein of PCV2 virions, such as figure 1 shown. Finally, the capsid protein (Cap protein) sequence encoded by PCV2 (GenBank: HM565924.1) was determined as a research template, and its amino acid sequence is shown in SEQ ID NO:4.

[0042] On the basis of the aforementioned PCV2-Cap protein analysis, combined with the characteristics that the host bacteria of the phage T7Select Phage Display System is Escherichia coli (E.coli BL5403), the nucleic acid sequence encoding PCV2-Cap was designed and optimized. The method is to ...

Embodiment 2

[0044] The preparation of embodiment 2 recombinant plasmids

[0045] The PCR product obtained in Example 1, that is, the recovered and purified DNA fragment was digested with E.coRI and HindIII, and the target fragment of about 720bp was recovered by electrophoresis detection, and the linear expression vector was also digested with E.coRI and HindIII. pET28a(+) ligation. The connection system is: 5.0 μl of target fragment, 1.0 μl of linear pET28a (+) vector, 1.00 μl of T4DNA ligase, 1.0 μl of 10×T4DNA ligase buffer, ddH 2 O2.0 μl. After ligation overnight at 4°C, the recombinant plasmid pET28a(+)-Cap was obtained.

Embodiment 3

[0046] Embodiment 3 Transformation and expression of protein

[0047] Mix 10 μl of the recombinant plasmid pET28a(+)-Cap prepared in Example 2 with 100 μl of BL21 competent cells, place in ice bath for 30 min, heat shock at 42°C for 90 s, quickly place in ice bath, rest for 2 min, add 800 μl of antibiotic-free LB medium After culturing on a shaker at 37°C at 150rpm for 45min, centrifuge at 5000rpm for 5min, discard the supernatant, leave 200μl to blow the bacteria evenly, and spread the LB plate containing kana, 100μg / ml, and incubate in an incubator at 37°C for 12-16h; A single white colony was selected every day, and a small amount of LB medium containing kana was used to amplify and culture; a small amount of plasmid was extracted by OMGA kit; it was identified by E.coRI and HindIII double enzyme digestion respectively. The enzyme digestion system was the same as before, and the results were as follows image 3 . The identified positive recombinant bacteria were named pET2...

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Abstract

The invention belongs to the biological field and particularly relates to preparation and application of recombinant porcine circovirus PCV2-Cap / Phage display particle. A preparation method of the capsid protein phage display particle of recombinant II porcine circovirus specifically comprises the following four steps: (1), optimizing and coding nucleotide of the capsid protein of the recombinant II porcine circovirus; (2), constructing a recombinant plasmid; (3), constructing a recombinant phage genome; and (4), preparing the capsid protein phage display particle of recombinant II porcine circovirus. An animal experiment proves that the recombinan display plasmid has good antigenicity, can stimulate a pig body to generate good humoral immunity and cellular immunity reaction, and can prevent and control II porcine circovirus infection; time needed for copying the process is short, production cost of the recombinant display particle is low, and product is convenient to use, simple and convenient to store and high in cloning yield.

Description

technical field [0001] The invention belongs to the biological field, in particular to the preparation and application of recombinant porcine circovirus PCV2-Cap / Phage display particles. Background technique [0002] Porcine circovirus (PCV) is a non-enveloped, CPE-free cell pollutant discovered by Tischer (1974) in the PK-15 cell line; it was identified as a single-stranded circular DNA virus in 1982. It is the smallest vertebrate virus discovered so far and belongs to the genus Circovirus in the family Circoviridae. Porcine circovirus is divided into two types: non-pathogenic porcine circovirus type 1 (PCV1) and pathogenic porcine circovirus type 2 (PCV2, the same below). [0003] Porcine circovirus disease (PCVD) is a weaning pig multisystemic wasting syndrome (Post weaning systemic wasting syndrome, PMWS) caused by PCV2 virus. It is the third largest infectious disease in the industry after swine fever and blue ear disease. Acute PCV2 infection damages the immune syst...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N7/01C12N15/34C12N15/66A61K39/12A61P31/20C12R1/92
Inventor 杨柳沈克飞郑华张邑帆付利芝谷山林熊仲良
Owner CHONGQING ACAD OF ANIMAL SCI
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