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Purifying method for hepatitis B e antigen

A purification method, hepatitis B technology, applied in the biological field, can solve problems such as cumbersome operation, and achieve the effect of simple and convenient operation and no impact on structure

Inactive Publication Date: 2014-06-25
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, GST (Glutathione S-transferase, glutathione sulfhydryl transferase) is used to fuse hepatitis B e antigen, but the relative molecular weight of GST is 26000, which has an impact on the protein structure of hepatitis B e antigen. The GST tag needs to be removed before applying the requirements, the operation is cumbersome, and the hepatitis B e antigen that removes the GST tag needs to be purified again to remove new impurities

Method used

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  • Purifying method for hepatitis B e antigen
  • Purifying method for hepatitis B e antigen
  • Purifying method for hepatitis B e antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Acquisition of target sequence of hepatitis B e antigen (HBeAg) gene

[0061] Using the pADR-1 plasmid containing the HbeAg gene stored in the laboratory as a template, using SEQ ID No.1 containing the XmaI restriction site as the upstream primer, and using SEQ ID No.2 containing the Not I restriction site as the downstream primer PCR amplification was performed to obtain the HBeAg gene. After recovery and purification of the amplified product, a hexahistidine-encoded gene was introduced at the 3' end of the HBeAg gene to form a recombinant HBeAg gene, and the recombinant HBeAg gene was digested with Xma I and Not I, and then inserted into pET-28a that was also digested In -c(+), the recombinant plasmid pET-28a-c(+)-HBeAg containing the recombinant HBeAg gene was obtained. The recombinant plasmids were transformed into Escherichia coli for preservation.

[0062] Induced expression of recombinant plasmids

[0063] The recombinant bacteria were cultured by shaking,...

Embodiment 2

[0075] Acquisition of target sequence of hepatitis B e antigen (HBeAg) gene

[0076] Using the pADR-1 plasmid containing the HbeAg gene stored in the laboratory as a template, using SEQ ID No.1 containing the XmaI restriction site as the upstream primer, and using SEQ ID No.2 containing the Not I restriction site as the downstream primer PCR amplification was performed to obtain the HBeAg gene. After the amplified product is recovered and purified, a hexahistidine-encoding gene is introduced at the 5' end of the HBeAg gene to form a recombinant HBeAg gene, and the recombinant HBeAg gene is digested with Xma I and Not I, and then inserted into pET-28a that has also undergone double digestion In -c(+), the recombinant plasmid pET-28a-c(+)-HBeAg containing the recombinant HBeAg gene was obtained. The recombinant plasmids were transformed into Escherichia coli for preservation.

[0077] Induced expression of recombinant plasmids

[0078] The recombinant bacteria were cultur...

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Abstract

A disclosed purifying method for hepatitis B e antigen comprises the following steps: transferring a recombinant plasmid of hepatitis B e antigen gene to a bacterium for obtaining a recombinant bacterium, wherein the 5' end and the 3' end of the hepatitis B e antigen gene are both connected with a poly-histidine encoding gene; adding an inducer for culturing the recombinant bacterium; performing lysis on the recombinant bacteria, and centrifuging the lysate; contacting an immobilized metal-chelated affinity chromatography medium with a supernatant obtained by centrifugation or with a precipitate solution prepared from a precipitate obtained by performing high-concentration denaturant dissolving and centrifugation under the condition that the poly-histidine fusion hepatitis B e antigen is allowed to combine the medium or to be absorbed by the medium; and selectively eluting poly-histidine fusion hepatitis B e antigen from the medium. According to the method, fusion tag technology and affinity chromatography are combined, the operation is simple and convenient; and by taking poly-histidine as a fusion tag, the structure of hepatitis B e antigen is not influenced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for purifying hepatitis B e antigen. Background technique [0002] The traditional hepatitis B e antigen mainly comes from the serum of infected persons. The source is difficult. The antigen titer of each batch is different, and the stability of the effect is poor. The production personnel have certain operational risks, and it is difficult to purify. [0003] With the development of molecular biology, there have been many methods for preparing hepatitis B e antigen by using genetic engineering technology. By constructing genetically engineered bacteria, a large amount of hepatitis B e antigen can be produced. However, the hepatitis B e antigen produced by these genetic engineering techniques has problems such as poor product titer and low immunogenicity, and in addition to hepatitis B e antigen, the genetically engineered bacteria product also includes various i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/51C07K14/02C07K1/22
Inventor 万晓春田永帅
Owner SHENZHEN INST OF ADVANCED TECH
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