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A kind of porcine circovirus type 2 antigen purification and concentration method

A porcine circovirus and antigen technology, applied in the field of biomedicine, can solve the problems of high cost of purification and concentration, poor immune protection effect of vaccines, and insignificant effect of antigen titer, so as to achieve high antigen recovery efficiency, reduce immune side effects, and improve Effects on Vaccine Safety

Active Publication Date: 2020-07-10
江苏省苏农科技术转移中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are still some important unresolved problems in the development and production of PCV2 vaccines in my country: 1. PCV2 has poor in vitro proliferation ability and low antigen titer, and the effect of improving antigen titer by optimizing culture conditions is not significant and limited; 2. Most of the PCV2 vaccines currently on the market are whole-virus inactivated vaccines, of which cell protein and bovine serum albumin account for more than 95%. Large-scale vaccination of such vaccines has certain side effects and will cause a waste of host immune resources , The immune protection effect of the vaccine is poor, etc.
[0004] At present, there is no relatively mature purification method for the production of circular vaccines in China. In the production of individual enterprises, membrane filtration clarification technology is used in conjunction with physical purification processes such as hollow fiber concentration technology for purification and concentration. Concentrate and remove part of the impurity protein, but there are defects such as complicated operation, high requirements for technical equipment, high cost of purification and concentration, low antigen recovery rate, and low concentration multiple. Therefore, it is urgently needed in the production of circovirus vaccines. A method capable of efficiently and cheaply purifying and concentrating vaccine-producing antigens to solve the problems of insufficient content and low purity of vaccine-producing antigens

Method used

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  • A kind of porcine circovirus type 2 antigen purification and concentration method
  • A kind of porcine circovirus type 2 antigen purification and concentration method
  • A kind of porcine circovirus type 2 antigen purification and concentration method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction and Identification of Recombinant Expression Plasmid pET-PL

[0028] Design the adapter protein, the amino acid sequence of the adapter protein is shown in SEQ ID NO: 1, the protein is a fusion protein, the fusion protein contains two functional recognition regions, which can specifically recognize the PCV2 antigen and the Lactococcus lactis backbone respectively, the linker The gene encoding the protein is shown in SEQ ID NO:2. In order to facilitate the identification of the adapter protein, a His tag protein (HHHHHH) was added to the carboxyl terminus of the sequence shown in SEQ ID NO: 1, and the His tag protein coding sequence was added before the stop codon of the adapter protein coding gene to obtain the sequence SEQ ID NO: 3, In addition, an Nde I restriction site was designed at the 5' end, and a HindIII restriction site was designed at the 3' end respectively, which were sent to GenScript for synthesis. Insert the synthesized gene fragm...

Embodiment 2

[0038] Example 2 Construction of Recombinant Expression Bacteria PL / BL21 and Expression of Adapter Protein

[0039] 1. Construction of recombinant expression strain PL / BL21

[0040]The recombinant plasmid pET-PL obtained in Example 1 was transformed into the prokaryotic expression strain BL21(DE3) to obtain the recombinant expression strain PL / BL21. At the same time, an empty pET32a plasmid was set as a control to transform BL21 (DE3) to obtain a control strain pET / BL21.

[0041] 2. Induced expression and identification of recombinant expression strain PL / BL21

[0042] (1) pick the single clones of bacterial strain PL / BL21 and control strain pET / BL21, respectively inoculate them into LB liquid medium containing 50 mg / mL ampicillin, and culture them overnight at 37°C and 200rmp to obtain mother liquor;

[0043] (2) Inoculate the mother liquor of each of the above strains into fresh LB liquid medium (containing 50 mg / mL ampicillin) at a ratio of 1:200, culture at 37°C and 180r...

Embodiment 3

[0053] Example 3 Adapter protein quantitative analysis

[0054] 1. Preparation of Purification Vector

[0055] Lactococcus lactis IL1403 (The Complete Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis ssp.lactisIL1403, Genome Res., 10.1101 / gr.169701) was cultured statically at 30°C with fresh GM17 medium, and the culture time was 16-18h. Centrifuge the culture solution at 8000rpm for 5min, collect the bacteria, wash the precipitate with PBS buffer once, add 0.1M hydrochloric acid to boil for 30min, centrifuge at 8000rpm for 5min, then wash the precipitate with PBS buffer for 3 times, and finally buffer with PBS solution to obtain the Lactococcus lactis skeleton, which is the purified carrier, and the hemocytocyte count of the purified carrier was obtained, and 2.5×10 9 A purified carrier is recorded as 1 unit.

[0056] 2. Protein Dissociation

[0057] The adapter protein solution was prepared according to the method in Example 2. In 2mL adapter protein soluti...

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Abstract

The invention provides a method for purifying and concentrating an antigen of PCV2 (porcine circovirus type 2) and relates to the technical field of biomedicine. The method for purifying and concentrating the antigen of PCV2 comprises the following steps: (1) adaptor protein with an amino acid sequence shown in SEQ ID NO:1 is added to the antigen of PCV2, mixed uniformly and incubated; (2) a purification carrier which is a lactococcus lactis skeleton is added, mixed uniformly and incubated; (3) centrifugation is performed, and precipitates are obtained. With the adoption of the method for purifying and concentrating the antigen of PCV2, a large amount of impure protein can be removed, the antigen recovery rate is high, the concentration multiple is high, requirements for equipment is low, operation is simple and the cost is low.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for purifying and concentrating porcine circovirus type 2 antigen. Background technique [0002] Porcine circovirus type 2 (PCV2) is the main pathogen that causes multisystemic wasting syndrome (PMWS) in weaned piglets. Since PMWS was first reported in Canada in 1991, the disease has spread to all parts of the world, and it is recognized as one of the important infectious diseases that endanger the swine industry in the world, and it is also very serious in our country. The use of vaccine immunization prevention is the key to solve the PCV2 epidemic problem. [0003] At present, there are still some important unresolved problems in the development and production of PCV2 vaccines in my country: 1. PCV2 has poor in vitro proliferation ability and low antigen titer, and the effect of improving antigen titer by optimizing culture conditions is not significant and limite...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/01C07K1/30C12N15/62C12N15/70A61K39/12A61P31/20
CPCA61K39/12C07K14/00C07K14/005C07K2319/00C12N2750/10034C12N2750/10051
Inventor 乔绪稳于晓明郑其升李鹏成陈瑾侯立婷张元鹏侯继波
Owner 江苏省苏农科技术转移中心有限公司
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