Hepatitis B virus antisense RNA expression vector targeting liver cancer and its construction process and use

A technology of hepatitis B virus and expression vector, applied in virus/bacteriophage, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of lack of specificity in transfection, difficult to achieve gene therapy, etc., and achieve good inhibitory effect and good inhibitory effect Effect

Inactive Publication Date: 2004-08-18
SHANDONG UNIV
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Problems solved by technology

[0009] Transfection of antisense RNA expression vector can make antisense RNA continuously expressed in host cells, but if the transfection lacks specificity, it is difficult to achieve the purpose of gene therapy

Method used

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  • Hepatitis B virus antisense RNA expression vector targeting liver cancer and its construction process and use
  • Hepatitis B virus antisense RNA expression vector targeting liver cancer and its construction process and use
  • Hepatitis B virus antisense RNA expression vector targeting liver cancer and its construction process and use

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Embodiment 1

[0163] Material

[0164] 1. Plasmids and strains: Plasmid pBR322-HBV is preserved in our laboratory, which contains 3.2kb HBV DNA of 2 copies of ayw subtype connected head to tail; vector pEBAF is provided by Dr. Yan Ziying, Institute of Virology, Chinese Academy of Preventive Medicine, with a total length of 15.5 Kb. Escherichia coli JM109 and DH5α are the strains preserved in this laboratory.

[0165] 2. Cell line and culture medium: The liver cancer cell line HepG2.2.15 integrates the HBV genome, can transcribe and translate HBV genes, and produce HBsAg, HBeAg, and Dane’s particles. The cell line is quoted from the Institute of Immunology, Shandong Academy of Medical Sciences. The human liver cancer cell line BEL7402 was passaged in this laboratory, expressing alpha-fetoprotein 200ng / ml. Human umbilical vein endothelial cells ECV-304 were donated by the Military Medical College, and the secretion of alpha-fetoprotein was not detected.

[0166] 3. Tool enzymes: Xbal, T4DN...

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Abstract

A hepatitis B virus antisense RNA expression vector targeted toward liver cancer is a gene therapeutic matter for hepatitis B virus related liver cell cancer. The present invention adopts EB virus expressing plasmid pEBAF as vector and positive liver cancer tissue expressing vector controlled by AFP promotor controlling and specifically expressed in AFP only to construct three kinds of HBV antisence RNA expression vectors pEBAF-as-preS2, pEBAF-as-preS1 and pEBAF-as-X, which contain HBV preS2, preS1 and X segment genes separately. Antisense RNA expression vector is prepared through amplifying a destination gene, designing, a primer, and a PCR amplification of HBpreS2, HBpreS1 and HBX genes. The antisense RNA expression vector is used for treating hepatitis B virus related liver cell cancer and hepatitis B virus infection.

Description

(1) Technical field [0001] The invention relates to a gene therapy drug for hepatitis B virus-related hepatocellular carcinoma, in particular to a hepatitis B virus antisense RNA expression vector targeting liver cancer and its construction method and application. (2) Background technology [0002] my country is a high-incidence area for hepatitis B virus (HBV) infection. It is estimated that there are about 1.2 million cases of acute viral hepatitis in my country every year; there are about 12 million cases of chronic hepatitis patients; about 300,000 cases of liver disease die each year, of which 50% are The vast majority of primary hepatocellular carcinoma is related to hepatitis B virus. According to the estimation of outpatient and hospitalization expenses of viral hepatitis in Shanghai in 1988, the annual direct medical expenses caused by viral hepatitis in my country amounted to 30-50 billion yuan. Although hepatitis B vaccination has been widely carried out in China,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/29A61K48/00C12N7/01C12N15/113C12N15/86C12Q1/68
Inventor 孙汶生马春红张利宁曹英林刘素侠王晓燕
Owner SHANDONG UNIV
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