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Preparation of pig secretory IgA secretory component and purification and renaturation method of pig secretory IgA secretory component

A secreted, positive technology, applied in the field of recombinant proteins, can solve the problems of low SC, high cost, cumbersome SC steps, etc., and achieve the effects of simple operation, increased concentration, and reduced production costs

Inactive Publication Date: 2019-01-11
广东海大集团股份有限公司畜牧水产研究中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, SC can be isolated and purified from colostrum, but the content of SC in porcine colostrum is low, and the steps of purifying SC are cumbersome and costly

Method used

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  • Preparation of pig secretory IgA secretory component and purification and renaturation method of pig secretory IgA secretory component
  • Preparation of pig secretory IgA secretory component and purification and renaturation method of pig secretory IgA secretory component
  • Preparation of pig secretory IgA secretory component and purification and renaturation method of pig secretory IgA secretory component

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Construction of recombinant plasmid for prokaryotic expression of SC protein

[0041] 1. Primer Design

[0042] Download the gene sequence of wild boar (Sus scrofa) polymeric immunoglobulin receptor (PIGR) from the NCBI website, and use bioinformatics software to analyze the signal peptide, transmembrane region and antigenicity of the protein. , designed a large number of primers for PCR amplification of SC gene, after a large number of screening, a set of primers SC-F and SC-R with strong sensitivity and specificity were selected, and BamHI and SalI were introduced into primers SC-F and SC-R respectively Restriction site, the sequence of the primer is as follows:

[0043] SC-F:5'-CCG GAATTC TCGGTGTCCATCAGATGCTACTA-3' (SEQ ID NO: 1);

[0044] SC-R:5'-ACGC GTC GAC CTGCAGGTACTCCACTCCCACTA-3' (SEQ ID NO: 2).

[0045] 2. Amplification of SC gene fragments

[0046] According to the instructions of the RNA extraction kit, the RNA of the porcine small intestine tissue w...

Embodiment 2

[0053] Fusion expression of SC protein

[0054] Method: The recombinant plasmids pET28a-SC and pGEX-4T-1-SC were respectively transformed into the expression strain BL21(DE3), and a single colony was picked in LB liquid medium. Detect the OD260 value in the LB liquid medium. If the OD260 is 0.4-0.6, add IPTG with a final concentration of 1mmol / L to induce. The induction condition is 16°C for 20 hours. identification of recombinant proteins.

[0055] Result: find that the protein expression of pET28a-SC (swimming lane 1) is higher than the protein expression of pGEX-4T-1-SC (swimming lane 3) ( image 3 ), the protein expression level was 10-20% higher; then the pET28a-SC plasmid with higher expression level was transformed into BL21(DE3) and Rosetta host bacteria respectively, and it was found that the protein expression level in Rosetta host bacteria (lane 3) was higher than BL21(DE3) host bacteria (lane 1), the protein expression level is 10-20% higher ( Figure 4 ).

[0...

Embodiment 3

[0058] Purification and renaturation of SC protein

[0059] 1. Purification of SC protein

[0060] The Rosetta host bacterium after the expression of the pET28a-SC plasmid of Example 2 was induced and expressed was washed 2-3 times with PBS, and the bacterial cells were broken by an ultra-high pressure continuous flow cell disruptor, centrifuged at 10000rpm for 15min, and the supernatant and the precipitate were separated, and SDS-PAGE was used to identify the recombinant protein SC mainly in the form of inclusion body. After the inclusion body is washed 3 times with the washing solution prepared by the present invention, most of the foreign proteins of the bacteria can be removed, which is better than that of the washing solution of the control group. Then the inclusion body protein was dissolved with 8M urea, and further purified by Ni column, it was found that there were almost no impurities and the bands were clearly visible, and the purification rate reached 90%, indicat...

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Abstract

The invention discloses preparation of a pig secretory IgA secretory component and a purification and renaturation method of the pig secretory IgA secretory component. SIgA is an important index for evaluating the mucosal immunity level of an organism, a secretory component SC is a specific component of an SIgA molecule, and the content of SIgA can be evaluated by virtue of SC proteins. Researching results on different prokaryotic expression vectors and host strains prove that by utilizing pET-28a prokaryotic expression vectors and Rosetta host strains, a large number of SC recombinant proteins can be expressed in vitro, after the SC recombinant proteins are purified and renaturated, the purities of the SC recombinant proteins are high, and the SC recombinant proteins can specifically bound with anti-nature SIgA polyclonal antibodies. The operation is simple, and a large number of the SC proteins with relatively high impurities can be acquired, so that the SC proteins have very good popularization and application values.

Description

technical field [0001] The invention belongs to the field of recombinant proteins, and relates to a method for preparing and purifying and refolding porcine secretory IgA secretion slices. Background technique [0002] Secretory immunoglobulin A (SIgA) is the main immunoglobulin on the mucosal surface. It can bind to pathogens on the mucosal surface such as the intestinal tract and respiratory tract to prevent pathogens from invading the body. SIgA is known as the first line of defense against mucosal infection. It exists in exocrine fluids such as the intestinal tract, respiratory tract, and urinary tract, as well as in colostrum, and the content in colostrum is the highest. Biologically, the level of SIgA is an indicator for evaluating the body's mucosal immunity. important indicators. [0003] Secretory component (SC) is an important part of SIgA molecule, which can be used to distinguish SIgA and IgA, and the antibody level of SIgA can be detected by SC. At present, SC...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/70C07K1/16C07K1/14
CPCC12N15/70C07K16/18
Inventor 朱利塞贾爱卿王贵平钱雪桥
Owner 广东海大集团股份有限公司畜牧水产研究中心
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