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Recombinant fusion protein and use thereof

A fusion protein and sequence technology, applied in the field of biomedicine, can solve the problems of disease progression, many adverse reactions, and unfixed course of treatment, etc.

Inactive Publication Date: 2015-02-11
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned interferon treatment course is relatively fixed, the HBeAg seroconversion rate is high, the curative effect is relatively long-lasting, and drug resistance does not occur; but there are many adverse reactions, and it is not suitable for high serum transaminase levels (such as higher than 10 times the upper limit of normal value) patients, and patients with decompensated liver cirrhosis[1-7]; oral administration of nucleoside (acid) analogues has a strong effect of inhibiting the virus, with few and mild adverse reactions, and can be used for the treatment of patients with decompensated liver cirrhosis. However, the course of treatment is not fixed, the HBeAg seroconversion rate is low, and long-term medication is required. After drug withdrawal, it is easy to relapse and the condition may worsen, and even life-threatening liver function decompensation occurs, and viral drug resistance mutations are prone to occur.
In summary, the current key hepatitis B treatment drugs are still unable to clear the HBV CCC-DNA in infected patients

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  • Recombinant fusion protein and use thereof
  • Recombinant fusion protein and use thereof
  • Recombinant fusion protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Taking the HBcAg-N149 fragment as an example to prepare the hepatitis B core antigen protein carrier or its truncated fragment

[0061] The gene fragment encoding hepatitis B core antigen HBcAg-N149 can be obtained from hepatitis B virus particles (HBV) by PCR method, and 1-149 amino acid residues are intercepted, and B is added to the 5' and 3' ends respectively. wxya I and P st Restriction site for I. The HBcAg-N149 gene fragment passed B wxya I and P st Restriction digestion of I, ligation and introduction of Escherichia coli expression plasmid Pcold IV, recombinant Escherichia coli expression plasmid into Escherichia coli expression host strain JM109. Put the positively identified recombinant colonies into 5 mL LB culture medium, and culture with shaking at 37°C until the culture medium reaches A 600 Up to 0.4-0.6. Take 2 mL of the culture solution and add it to 200 mL of ampicillin-based LB culture solution, shake and culture for 3 h, then add IP...

Embodiment 2

[0062] Example 2 Insert B cell epitopes into reasonable sites of HBcAg-N149 to form fusion proteins as an example to prepare recombinant fusion proteins of HBcAg or truncated fragments carrying HBV therapeutic polypeptide epitopes

[0063] Insert the gene sequence encoding the 14th-24th amino acid sequence (DPRVRGLYFPA) derived from HBV Pre-S2 into the 78-79th amino acid of the HBcAg-N149 fragment by overlapping PCR, and add B to the 5' and 3' ends respectively wxya I and P st Restriction site for I. The HBcAg-N149 gene fragment passed B wxya I and P st Restriction digestion of I, ligation and introduction of Escherichia coli expression plasmid Pcold IV, recombinant Escherichia coli expression plasmid into Escherichia coli expression host strain JM109. Put the positively identified recombinant colonies into 5 mL LB culture medium, and culture with shaking at 37°C until the culture medium reaches A 600 Up to 0.4-0.6. Take 2 mL of the culture solution and add it to 200 ...

Embodiment 3

[0065] Taking recombinant HBcAg-N149 fragment and recombinant fusion protein HBcAg-N149 / preS2 as an example to illustrate the experiment of vaccine-induced HBV-specific immune response

[0066] Three experimental groups were set up, namely the normal saline group, the HBcAg-N149 group, and the recombinant fusion protein HBcAg-N149 / preS2 group; tested in 6 female BALB / c mice (each group), each mouse was at 0, On days 21 and 42, 0.1-100 μg fusion protein and 10 μg aluminum salt adjuvant were subcutaneously injected.

[0067] One week after the second immunization, the antibody against the preS2-B epitope was detected, and the results were as follows image 3 shown, there are more than 10 5 Anti-preS2-B epitope antibody titer, and more than 10 3 The titer of the antibody against HBc showed a dose-dependent relationship; HBcAg-N149 could not induce antibodies against the preS2-B epitope; one week after the third immunization, the cellular immune response (IL4 and IFNγ) in the sp...

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Abstract

The invention belongs to the biological medicine field, relates to recombinant fusion protein and use thereof, and in particular relates to recombinant fusion protein carrying hepatitis B virus therapeutic antigen epitopes which are inserted into hepatitis B core antigen protein particles or truncated fragments and use thereof. The recombinant fusion protein contains multiple epitope antigens of hepatitis B virus (HBV) and other immune stimulating epitope antigens and hepatitis B core antigen virus-like particles or truncated fragments thereof for preparation of chimeric antigen, the multiple antigen epitopes can be inserted into same or different sites of hepatitis B virus core antigen HBc or truncated fragments thereof in the manner of single epitope or multi epitope combination, and by combination with different adjuvants, HBV specific humoral and cellular immune functions can be strengthened.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a recombinant fusion protein and its application, in particular to a recombinant fusion protein carrying a hepatitis B virus therapeutic antigen epitope inserted into a hepatitis B core antigen protein particle or a truncated fragment and its application. Background technique [0002] According to statistics, as of 2010, the prevalence rate of HBsAg in the whole country was 7.18%, compared with 11% in the past, and the downward trend is obvious, indicating that the relevant prevention and control measures currently adopted are effective. Due to the huge population base, there are still about 93 million hepatitis B virus carriers in China, of which about 20-30 million are chronic hepatitis B patients. Studies have shown that chronic hepatitis caused by hepatitis B virus infection is closely related to liver failure, liver cirrhosis and primary liver cancer. For example, more than 90% of li...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K39/29A61K39/39A61P31/20
Inventor 史训龙周珮鞠佃文周伟黄海冯美卿朱海燕叶丽
Owner FUDAN UNIV
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