Expression vector PVX-6His-CTBt-Bt for producing multi-epitope vaccine of hepatitis c virus

A technology of pvx-6his-ctbt-bt and 6his-ctbt-bt, which is applied in the field of expression vectors for the production of hepatitis C (hepatitis C) virus multi-epitope vaccines

Inactive Publication Date: 2015-02-18
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, to date there is no vaccin

Method used

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  • Expression vector PVX-6His-CTBt-Bt for producing multi-epitope vaccine of hepatitis c virus
  • Expression vector PVX-6His-CTBt-Bt for producing multi-epitope vaccine of hepatitis c virus
  • Expression vector PVX-6His-CTBt-Bt for producing multi-epitope vaccine of hepatitis c virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The construction and identification of PVX-6His-CTBt-Bt carrier, its method is as follows:

[0034] (1) Vaccine amino acid composition design:

[0035] Amino acid composition design of hepatitis C hepatitis virus multi-epitope vaccine: 6 histidines from N-terminus to C-terminus (to facilitate vaccine purification), oral immune adjuvant cholera toxin B subunit CTB (enhance the oral immune effect of the vaccine), Six B-cell neutralizing epitopes of Bt (E1 313-327aa -E2 396-424aa -E2 438-447aa -E2 523-540aa -E2 610-627aa -E2 631-648aa ). Each epitope is separated by a glycine and a serine residue).

[0036] The amino acid sequence of the hepatitis C virus vaccine 6His-CTBt-Bt designed according to the above requirements is:

[0037] His His His His His His His Met Ile Lys Leu Lys Phe Gly Val Phe Phe

[0038] 1 5 10 15

[0039]Thr Val Leu Leu Ser Ser Ala Tyr Ala His Gly Thr Pro Gln Asn Ile

[0040] 20 25 30

[0041] Thr Asp Leu Cys Ala Glu Tyr His Asn Thr Gln ...

Embodiment 2

[0150] Vaccine expression and Western blot identification in tobacco, its method is as follows:

[0151]Corundum worn tobacco leaves were inoculated with the vaccine expression vector PVX-6His-CTBt-Bt. At the same time, potato X virus PVX201 (negative control) and PVX204 (carrying GFP gene) were inoculated with tobacco leaves as controls, and the amount of virus vector inoculated on each leaf was 2 -4μg. To verify the expression of GFP protein, to optimize the effect of virus inoculation and replication, to prepare the protein extract of tobacco, and to use mouse-derived 6×His Tag antibody (LanPower?) and CTB antibody (antibodies-online Inc), respectively, for Western blot Detection of vaccine expression. Ni column purification of expressed vaccine protein 6His-CTBt-Bt.

[0152] According to the experimental method described above, the recombinant expression vector PVX-6His-CTBt-Bt was inoculated into tobacco. At the same time, the positive control PVX204 (expressing GFP gene...

Embodiment 3

[0154] Animal experiments are used to determine the immune characteristics and safety of vaccines, and the methods are as follows:

[0155] Four-week-old BALB / c mice were orally immunized with the vaccine protein purified by Ni column three times (immunization time was 1, 14 and 28 days), and each time each mouse was immunized with 10 μg of vaccine protein. Rats immunized with tobacco leaf protein extract (10 μg) without vaccine served as controls. Three mice were immunized in the experimental group and the control group respectively. Blood was collected at different times, serially diluted 2 times, and antibody titers were determined at different periods (1, 14, 28, 56 and 70 days) by ELISA. Under the condition that the OD value (490nm) of the positive serum is at least 2 times greater than the OD value of the negative control (rat serum before vaccine immunization and mouse serum not immunized with vaccine), the maximum dilution of positive serum is the titer of this antibo...

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Abstract

The invention discloses an expression vector PVX-6His-CTBt-Bt for producing a multi-epitope vaccine of hepatitis c virus. The multi-epitope vaccine 6His-CTBt-Bt of hepatitis c virus expressed by the vector comprises a histidine tag, an orally-taken immunologic adjuvant cholera toxin B subunit (CTB) and six B cellular antigen epitopes Bp of refractory hepatitis c virus genetype 1. To facilitate high-efficiency expression of the vaccine in tobacco, the 6His-CTBt-Bt gene optimized by an artificially synthesized codon is cloned to a high-efficiency plant virus expression vector potato X virus vector PVX201 to obtain the recombinant expression vector PVX-6His-CTBt-Bt of the multi-epitope vaccine 6His-CTBt-Bt of the hepatitis c virus; the expression vector is inoculated to the tobacco; the tobacco is regarded as a reactor to produce the anti-hepatitis c virus vaccine which aims to the refractory hepatitis c virus, is easy to purify, and can achieve a good orally-taken immune effect and resist against virus infection in vitro.

Description

technical field [0001] The invention relates to the field of research and development of antiviral vaccines, in particular to an expression vector PVX-6His-CTBt-Bt for producing hepatitis C (hepatitis C) virus multi-epitope vaccines. Background technique [0002] Hepatitis C virus is the pathogenic factor of hepatitis C. The total number of people infected by the virus exceeds 170 million in the world, and the number of infected people in my country exceeds 40 million. Most people with HCV infection are persistent, leading to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. According to reports, 40% of global chronic liver diseases and 80% of hepatocellular carcinoma are related to hepatitis C virus, and the annual global cost of hepatitis C treatment exceeds 100 billion US dollars. Existing therapeutic drugs are only effective for some hepatitis C virus-infected patients, and the treatment time is long, the cost is high and there are side effects. Vaccines are ...

Claims

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Application Information

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IPC IPC(8): A61K39/29A61K39/39A61P31/14
Inventor 孔令保黄明洁郭韫丽
Owner JIANGXI AGRICULTURAL UNIVERSITY
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