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Preparation method of Cap antigen for detecting porcine circovirus 2 type antibody

A technology of porcine circovirus and type 2 antibody, which is applied in the field of preparation of soluble Cap antigen, achieving the effect of high purity and reliable technical support

Inactive Publication Date: 2008-01-09
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The technical problem to be solved in the present invention is to express and produce soluble rCap protein with good biological activity in Escherichia coli, and to achieve the purpose of reducing the formation of inclusion bodies by changing the conventional induced expression conditions and medium components, avoiding complex denaturation, renaturation process, and then developed a serum antibody detection method and device with simple operation, reliable results, high sensitivity and good specificity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The gene encoding PCV2 ORF2 was cloned by PCR and the recombinant expression vector rCap was constructed as described above.

[0043] The recombinant bacteria were inoculated into 1L cLB with 1% inoculum (Kan + 80mg / L, 2 L Erlenmeyer flask) liquid medium, wherein the composition and preparation method of cLB liquid medium are as follows: peptone 8g; sodium chloride 8g; yeast extract 4g, add deionized water to 1L, adjust pH with NaOH =7.0, 37°C enrichment for 3 hours, measured OD 600 =0.5~0.6, add a certain amount of IPTG to make the final concentration 0.1mol / L, induce (first induction) at 37°C and 180r / min for 4h, then continue to induce (second induction) at 16°C for 18h; 5000r / min Centrifuge for 10 min, collect the bacteria with 1×PBS buffer, oscillate and mix; ultrasonically break the bacteria (work for 4 s, intermittent for 6 s, 50 cycles, ultrasonic three times, temperature controlled at 4°C); centrifuge at 4°C, 15,000 r / min for 30 min. 15% SDS-PAGE protein gel ...

Embodiment 2

[0047] Repeat the method of Example 1, except that the composition and preparation of the cLB liquid medium are different, as follows: 6 g of peptone; 9 g of sodium chloride; 3 g of yeast extract, add deionized water to 1 L, and adjust the pH to 7.0 with NaOH.

[0048] 15% SDS-PAGE protein gel electrophoresis showed that the target protein existed in the supernatant in the form of soluble matter, with a size of about 32 KDa.

[0049] The soluble rCap protein was characterized by Western blotting and indirect ELISA as described above, and the results are shown in Table 1.

Embodiment 3

[0051] Repeat the method of Example 1, except that the composition and preparation of the cLB liquid medium are different, as follows: 9 g of peptone; 6 g of sodium chloride; 2 g of yeast extract, add deionized water to 1 L, and adjust the pH to 7.0 with NaOH.

[0052] 15% SDS-PAGE protein gel electrophoresis showed that the target protein existed in the supernatant in the form of soluble matter, with a size of about 32 KDa.

[0053] The soluble rCap protein was characterized by Western blotting and indirect ELISA as described above, and the results are shown in Table 1.

[0054] Table 1

[0055]

Example

Peptone

Sodium chloride

Yeast extract

protein concentration

Western

blotting *

OD450

1

8g

8g

4g

45.23mg / ml

Have

have

2

6g

9g

3g

none

Have

have

3

9g

6g

2g

none

Have ...

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PUM

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Abstract

A preparation method of the Cap antigen of testing pig cirque virus two type antibody, the method includes steps as follow: (1)PCR clone coding gene of PCV2 ORF2; (2)designing and reforming the expression vector rCap; (3)in the culture medium of improved LB liquid, 37deg induction orderly and low temperature long-playing induction rCap expression; (4) sublimating rCap protein; of which the culture medium of improved LB liquid containing 6-9g / L peptone,6-9g / L sodium chloride and 2-4g / L yeast extract; taking the inductor IPTG to induce rCap expression, the finally thickness of it is 0.05-0.2 mol / L; the first inductive temperature of inducing the rCap expression is 37deg, the second inductive temperature of inducing the rCap expression is 16-20deg; the first inducing time is 3-6 hours for induction rCap expression, the first inducing time is 12-20 hours. The method accelerates rCap to bring solubility rCap proteinin the Escherichia coli, reducing the form of occlusion body, avoiding the process of the complicated denaturation and renaturation, at the same time, the biology activity of solubility rCap protein which gets by separating and sublimating is close to PCV2 virus native protein.

Description

technical field [0001] The present invention relates to a preparation method of Cap antigen, more specifically, relates to a preparation method of soluble Cap antigen which can be used for detecting porcine circovirus type 2 antibody. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) belongs to circoviridae (circoviridae) genus, is a non-enveloped single-stranded circular negative-sense DNA virus, and is one of the smallest animal viruses known so far. Tischer (1974) first discovered PCV in the porcine kidney cell line PK-15. According to the antigenicity and genetic composition of PCV, it is divided into two genotypes (or serotypes), namely PCV1 and PCV2. PCV1 is non-pathogenic and widely exists in pigs and pig-derived cell lines; PCV2 is pathogenic and is one of the main pathogens causing Post-weaning multisystemic wasting syndrome (PWMS) in weaned piglets. [0003] PCV2 is one of the newly discovered infectious diseases of pigs in recent years. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/53
Inventor 刘湘涛尹双辉尚佑军孙世琪刘艳红
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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