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Preparation method of human Ro52 recombinant protein

A recombinant protein and recombinant vector technology, applied in the field of preparation of human Ro52 recombinant protein, can solve the problems of complex operation, high cost, long cycle, etc., and achieve the effects of high purity, low cost and high yield

Inactive Publication Date: 2018-05-11
SUZHOU HAOOUBO BIOPHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It uses a eukaryotic expression vector for the preparation of Ro52 protein. This method has problems such as complicated operation, long cycle, high cost, and low yield.

Method used

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  • Preparation method of human Ro52 recombinant protein
  • Preparation method of human Ro52 recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Artificial synthesis of human Ro52 gene

[0041] According to the published DNA sequence of human Homo sapiens tripartite motif containing 21 (TRIM21) in GenBank (GenBank sequence accession number: BC010861), the full-length sequence of the gene is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO.2. The full-length Ro52 gene was directly synthesized artificially after codon optimization in the prokaryotic expression system, and was subcloned into the expression vector pET-28a (+).

Embodiment 2

[0042] Example 2: Preparation of expression strain BL21(DE3)-pET28a (+)-Ro52

[0043] 1. Take out the prepared Escherichia coli BL21(DE3) competent cells and put them in an ice bath until they melt.

[0044] . Add 10 µL of the above recombinant plasmid pET28a (+)-Ro52, ddH to every 100 mL of competent cells 2 O 1 µL (negative control), gently aspirate and beat evenly with a pipette, and place on ice for 30 min.

[0045] . Place the centrifuge tube in a 42°C water bath and heat shock for 90 s.

[0046] . Quickly transfer the centrifuge tube to an ice bath and let it stand for 2 min.

[0047] . Add 500 µL of LB medium to each tube, and incubate at 37°C for 1 h on a shaker at 180 rpm to recover the bacteria. Centrifuge at 4000 rpm for 3 min, discard about 500 µL of supernatant, and resuspend the cells.

[0048] . Spread 100 µL of the recombinant plasmid transformation product and 100 µL of the negative control evenly on the LB plate containing 100 µg / mL Amp (ampicillin)...

Embodiment 3

[0050] Example 3: Human Ro52 Gene Fusion Expression

[0051] Pick a single colony into 6 mL of LB containing 100 mg / mL Amp, and culture overnight at 37°C and 250 rpm; take 5 mL of bacterial liquid and add it to 500 mL of LB containing 100 mg / mL Amp, and culture at 37°C and 250 rpm. When OD600=0.6, a final concentration of 1 mMIPTG (isopropylthiogalactopyranoside) was added, and induction was continued at 37°C and 180 rpm for 4 h, and centrifuged at 10,000 rpm for 5 min to collect the bacteria.

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Abstract

The invention relates to a preparation method of a human Ro52 recombinant protein. The preparation method comprises the following steps: (1), artificially synthesizing a human Ro52 gene and subcloningthe gene to a prokaryotic expression vector to obtain a recombinant vector; (2), enabling the recombinant vector obtained in step (1) to enter a prokaryotic expression system by transforming to obtain a transformant; (3) culturing the transformant obtained in step (2) in a culture medium to obtain expression bacteria; (4) obtaining the human Ro52 recombinant protein from the expression bacteria obtained in step (3) by affinity chromatography. By adopting the prokaryotic expression system and controlling process parameters, the preparation method of the human Ro52 recombinant protein is simple, easy to operate, low in cost and high in yield (2.95 mg of the human Ro52 recombinant protein can be obtained under 1 L of induced expression), and purity of the human Ro52 recombinant protein is high (higher than 90%).

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and bioengineering, and in particular relates to a preparation method of human Ro52 recombinant protein. Background technique [0002] When using soluble tissue antigens and Sjogren's syndrome (Sjfgren's syndrome) patient serum for immunodiffusion experiments, two specific antigens were found, named Sjögren's syndrome type A antigen and B type antigen, and then found that Sjögren's syndrome type A antigen ( SSA) consists of two proteins, named Ro52 and Ro60. Ro52 is the target antigen of many autoimmune diseases, such as systemic lupus erythematosus, Sjogren's syndrome, systemic sclerosis and dermatomyositis, etc. Anti-Ro52 antibody is also an important disease cause of congenital heart block in newborns. A number of studies have shown that there are high titers of anti-Ro52 antibodies in the serum of patients with Sjogren's syndrome, so the development of Ro / SSA antibody detection k...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/12
CPCC07K14/4713C12N15/70C12N2800/22
Inventor 李庆春周延庆钱林杨阳陈瑞勤
Owner SUZHOU HAOOUBO BIOPHARML
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