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Preparation method of phage display-expressing circovirus antigen vaccine

A phage display, circovirus technology, applied in the direction of viral antigen components, antiviral immunoglobulins, viral peptides, etc., can solve the problems of complex methods, little effect and high cost, and achieve easy preparation, good protection effect, The effect of low production cost

Inactive Publication Date: 2015-04-01
SHANDONG SINDER TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current treatment methods for PCV2 are complex and costly, with little benefit from technical effects

Method used

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  • Preparation method of phage display-expressing circovirus antigen vaccine
  • Preparation method of phage display-expressing circovirus antigen vaccine
  • Preparation method of phage display-expressing circovirus antigen vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Acquisition of porcine circovirus type Ⅱ ORF2 gene and preparation of polyclonal antibody for immune screening

[0022] 1 Acquisition of porcine circovirus type Ⅱ ORF2 gene.

[0023] Search the ORF2 sequence of PCV2 on GENBANK, search for the open reading frame according to the obtained sequence, and design the following primers according to the sequence of the open reading frame:

[0024] Upstream primer 5'-CGGGTACCGCCACCATGACGTATCCAAGGAGGC-3'

[0025] Downstream primer 5'-GCGGATCCTCACTTAGGGTTAAGTGGGGGG-3'

[0026] Extract the mRNA of porcine circovirus type Ⅱ virus culture solution, reverse transcribe it into cDNA, and use this as a template to amplify the ORF2 fragment by PCR. The amplification results of the target gene are as follows: figure 1 As shown, 704bp is the target band, connected to the PMD-18T vector, and the characteristics of the gene fragment obtained by sequencing are as follows:

[0027] The length of the ORF is 704bp, the start codon is...

Embodiment 2

[0037] Example 2 Insertion of porcine circovirus type Ⅱ ORF2 gene into T4 phage

[0038] After the porcine circovirus type II ORF2 gene fragment was ligated and inserted into the pMD-18T vector, it was digested with EcoR Ⅰ and hindIII to recover the digested fragment, and the T4 phage expression plasmid was digested with EcoRI and hindIII. The digested fragment was recovered, T4 ligase was used to connect the gene fragment to the SOC site of T4 phage, and the ligated recombinant T4 phage was used to infect X-Blue ST1 CCTCC M 2014397 host cells.

Embodiment 3

[0039] Example 3 Immune Screening

[0040] 1 Preparation of reagents

[0041] LB Tetracycline: add 10mg tetracycline to every 1mL of distilled water, dissolve and sterilize with a 0.22μm filter, store at -20°C.

[0042] Maltose-supplemented LB medium: 1L of LB medium, 0.2Mpa at 121°C, 20min, add 1M MgSO4 (10mL) and 2M maltose (3mL) filtered through a 0.22μm filter.

[0043] NZY agar: NaCl 5g, MgSO 4 .7H 2 O 2g, yeast extract 5g, hydrolyzed casein (NZ Qmine) 10g, agar 15g. Add deionized water to 1L, adjust pH=7.5, 121℃0.2Mpa, 20min, after cooling down to room temperature, pour into a petri dish.

[0044] NZY medium: NaCl 5g, MgSO 4 .7H 2 O 2g, yeast extract 5g, NZ Qmine 10g. Add deionized water to 1L, adjust pH=7.5, 121℃0.2Mpa, 20min.

[0045] NYZ top agar: NaCl 5.8g, MgSO 4 .7H 2 O 2g, 1M Tris-HCl (pH=7.5) 50.0ml, 2% (w / v) gelatin 0.5ml. Prepare 1L of NYZ sterilized medium, add 0.7% (w / v) agarose, 0.2Mpa, 0.2Mpa at 121°C, and sterilize for 20min.

[0046] SM buffer...

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Abstract

The invention provides a preparation method of a phage display-expressing circovirus antigen vaccine. The preparation method comprises the following steps: extracting porcine circovirus II type (PCV2) RNA, carrying out inverse transcription, carrying out a polymerase chain reaction (PCR) for amplification so as to obtain porcine II type circovirus ORF2 gene's open reading frame fragments, directionally inserting the fragments into a T4 phage; obtaining a T4 phage with the ORF2 gene by an immunoscreening method; purifying and infecting X-Blue ST1(CCTCC M 2014397) host cell again; carrying out fermental cultivation to obtain the X-Blue ST1 host cell, and carrying out inducible expression and inactivation and taking a culture as an antigen; carrying out ELISA calibration of antigen valence, and carrying out double dilution on the antigen with normal saline to the final concentration of 107U / ml; and a vaccine adjuvant is added according to the ratio of 1:2.5, so as to obtain the vaccine. According to the invention, the phage is utilized to express the porcine circovirus II type antigen and the vaccine is prepared. Thus, the virulence enhancement risk of an attenuated vaccine is avoided. In addition, fermental cultivation is beneficial to large-scale industrial production. Cost of the vaccine provided by the invention is far lower than that of a vaccine produced by a cell culture method.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of a circovirus antigen vaccine expressed by phage display. Background technique [0002] Porcine circovirus infection is an acute infectious disease of pigs caused by circovirus. The main features are emaciation, decreased physical fitness, anemia, jaundice, diarrhea, stunted growth, dyspnea, and reproductive impairment in sows. with extensive pathology. According to the different serotypes, it can be divided into PCV1 and PCV2. Among them, PCV2 is the most serious infection reported in my country. Currently, the methods for treating PCV2 are complex and costly, and the technical effects and gains are minimal. Contents of the invention [0003] The purpose of the present invention is to aim at the deficiencies of the prior art, and provide a preparation method of a circovirus antigen vaccine expressed by phage display, so as to control the infe...

Claims

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Application Information

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IPC IPC(8): A61K39/12A61P31/20C12N15/70C07K14/01C07K16/08C07K16/06
Inventor 李明义高江明刘阳李晓林单学强张伦冯晶晶李佳棋孙化露葛栋郭春丽
Owner SHANDONG SINDER TECH
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