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Liver targeting cell-penetrating antiviral fusion polypeptide as well as coding gene and application thereof

A fusion of polypeptide and anti-virus technology, applied in the direction of anti-viral agents, peptides, desipeptides, etc., can solve the problems of clearing the body's immune system, difficult to achieve precise cell targeting, etc., to achieve precise active targeting, accurate positioning, and good application foreground effect

Inactive Publication Date: 2012-09-19
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the influence of various factors such as the receptor level and the type, size and specificity of the carrier, although the current targeting research has a certain degree of targeting, it is still difficult to achieve precise targeting at the cellular level, and it is easy to be immune. Insufficient system cleanup etc.

Method used

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  • Liver targeting cell-penetrating antiviral fusion polypeptide as well as coding gene and application thereof
  • Liver targeting cell-penetrating antiviral fusion polypeptide as well as coding gene and application thereof
  • Liver targeting cell-penetrating antiviral fusion polypeptide as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 SOE-PCR method to construct fusion gene

[0030] 1. Primer design ( figure 1 )

[0031] Four oligonucleotide primers were designed with Primer Premier 5.0 biological software, and the primer sequences are shown in SEQ ID NO: 3-6. Wherein H1 and M1 are primers in the same direction as the target gene, H2 and M2 are primers in the complementary part to the target gene, and M1 and M2 are used to amplify the MDC fragment from the pMD 20-T / MDC plasmid. There are 16 bp overlapping regions between H1 and H2, H2 and M1 respectively. Included in the upstream primer H1 Kpn I enzyme cleavage site and protease cleavage site, the stop codon and the downstream primer M2 are designed Hind Ⅲ Restriction site, the position of each primer in the target gene is as follows: figure 1 shown.

[0032] 2. The SOE-PCR method is used to construct a fusion gene containing the nucleotide sequence shown in SEQ ID NO.2, divide the entire sequence into two segments (HM1, HM2),...

Embodiment 2

[0049] Example 2 Solid-phase chemical synthesis of fusion polypeptide, anti-HBV effect and liver-targeted transmembrane research

[0050] 1. Solid-phase chemical synthesis of novel liver-targeting membrane-penetrating antiviral fusion polypeptide:

[0051] According to the sequence of amino acid residues of SEQ ID NO: 1, Beijing Zhongke Yaguang Co., Ltd. was entrusted to synthesize the polypeptide, which was purified to a purity of 95%.

[0052] 2. Research on the anti-HBV effect of the fusion polypeptide: HepG2.2.15 cells were used as the HBV-infected cell model, and HepG 2.2.15 cells were cultured with culture medium containing different concentrations of drugs, and the culture medium containing drugs was replaced every 3 days. 3, 6, 9 day supernatants were stored at -80°C. The content of HBsAg and HBeAg in the cell supernatant was detected by ELISA, and the concentration of HBV DNA was determined by Real-time PCR. The results showed that the fusion polypeptide could ...

Embodiment 3

[0055] Example 3 Recombinant expression of novel liver-targeting membrane-penetrating antiviral fusion polypeptide

[0056] In this example, the pET expression system of Novagen was used to express the fusion gene HTPP-MDC, the pET32a(+) plasmid was used as the expression vector, and the host cell was Escherichia coli.

[0057] 1 Construction of fusion gene expression vector

[0058] Recombinant plasmid pMD 20-T / HTPP-MDC and expression vector pET32a(+) were respectively treated with restriction endonuclease Kpn I and Hind Ⅲ Carry out double enzyme digestion, then connect overnight with T4 ligase, and transform the ligated product E. coli BL21, the transformed strain was spread on a plate containing ampicillin, and after culturing for 16-18 hours, a single colony was picked and cultured for bacterial liquid PCR, enzyme digestion identification and DNA sequencing verification, the results showed that the fusion gene expression plasmid pET32a / HTPP-MDC was constructed s...

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PUM

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Abstract

The invention discloses liver targeting trans-membrane antiviral fusion polypeptide as well as a coding gene and an application thereof. The fusion polypeptide is polypeptide consisting of an amino acid residue sequence shown in SEQ IDNO: 1. The invention further discloses a coding gene of liver targeting cell-penetrating peptide-cecropin fusion peptide, a nucleotide sequence of the coding gene is SEQ IDNO: 2. The fusion polypeptide has the following advantages: 1) the positioning is accurate, and the combination is highly-efficient; 2) competitive blocking and intracellular targeting killing are performed; and 3) the toxic and side effects are small. The liver targeting trans-membrane antiviral fusion polypeptide provided by the invention has a larger practical significance and a wide application prospect in the preparation field of anti-HBV (Hepatitis B Virus) drugs.

Description

Technical field [0001] The present invention involves the field of biotechnology, and specifically involves an antiviral fusion peptide peptide and its coding genes and applications with liver -targeted membrane effects. Background technique [0002] Hepatitis B Virus (HBV) is hepatitis. More than 350 million people in the world are infected with HBV. my country is an HBV high pop area. According to the national population BV epidemiological survey, the population HBSAG carrying rate reaches 7.18%. About 93 million people.HBV infection can lead to the occurrence of acute and chronic hepatitis B. It is one of the important factors that cause liver cirrhosis and hepatocytoma. They died of as high as 1 million people in hepatitis B related diseases each year.A common cause of death.A large number of clinical studies have proved that the continuous activity and development of HBV's continuous existence and continuous replication caused by liver lesions is the root cause of the progre...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21A61K38/16A61P31/12
Inventor 朱家勇卢雪梅金小宝黄演婷汪洁
Owner GUANGDONG PHARMA UNIV
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