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Fusion protein of liver targeting peptide and human interferon a2b and its preparation method and application

A fusion protein and liver-targeting technology, applied in the biological field, can solve problems such as clearance, difficult to achieve precise cell targeting, and easy immune system of the body, and achieve the effects of improving specificity, precise and active targeted clearance, and reducing systemic dosage

Active Publication Date: 2011-12-07
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 type, size and specificity of the carrier, although the current IFN 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 cleared by the body's immune system. Not enough

Method used

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  • Fusion protein of liver targeting peptide and human interferon a2b and its preparation method and application
  • Fusion protein of liver targeting peptide and human interferon a2b and its preparation method and application
  • Fusion protein of liver targeting peptide and human interferon a2b and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Gene modification, fusion gene IFN-CSP recombination and construction of expression plasmid

[0036] In this example, Novagen's pET expression system was used to express the fusion gene IFN-CSP, the pET32a(+) plasmid was used as the expression vector, and the host cell was Escherichia coli.

[0037] 1 Gene modification and fusion gene IFN-CSP recombination ( Figure 1-2 )

[0038] According to the amino acid sequence of human IFNa2b (NP_000596) and the amino acid sequence of Plasmodium falciparum CSP I-plus (CAA33421) published by NCBI, using E. coli preferred codons, computer-aided design of the nucleus of the liver-targeted interferon IFN-CSP gene Nucleotide sequence. The whole gene was divided into 16 overlapping oligonucleotide fragments, and primers P1-P16 were designed with Primer Premier 5.0 biological software. The primer sequences are shown in SEQ ID NO: 3-19. Where upstream P1 is introduced Kpn I restriction site, downstream P16 introduces a stop codon...

Embodiment 2

[0049] Example 2 Preparation of fusion protein IFN-CSP ( Figure 4 )

[0050] Transform the IFN-CSP / pET32a plasmid into the prepared expression host bacteria E.coli BL21 (DE3) competent cells, after ampicillin resistance screening, pick IFN-CSP / pET32a positive single colony into 5ml LB liquid medium (1wt% peptone, 0.5wt% yeast extract powder, 85 mmol / L Sodium chloride), 37℃, 220rpm shaking for 8~12h, and then this bacterial solution was inoculated into fresh LB liquid medium at a ratio of 1:100, 37℃, 220rpm shaking to OD600 about 0.6, add the final IPTG at a concentration of 1.0 mmol / L was induced for 4-6 hours. Collect the bacterial cells by centrifugation and add 1×SDS-PAGE Buffer (50mM Tris-HCl pH6.8, 100mM DTT, 2% SDS, 0.1wt% bromophenol blue, 10wt% glycerol), boil for 5min, centrifuge, and take the supernatant for sample. Make 6 vol% concentrated gel, 15 vol% separation gel, electrophoresis voltage: concentrated gel 80V, separation gel 120V. After the electrophoresis, t...

Embodiment 3

[0052] Example 3 In vitro pharmacodynamics and specific targeting effect on liver cells of the fusion protein IFN-CSP of the present invention

[0053] (1) In vitro pharmacodynamic study of IFN-CSP: HepG2.2.15 cells were used as the HBV infected cell model, and HepG 2.2.15 cells were cultured in culture medium containing different CSP-IFN concentration gradients, and the drug-containing culture was replaced every 3 days The supernatant was collected on the 3rd, 6th, and 9th day. HBsAg and HBeAg in the cell supernatant were detected by ELISA, and the A value was measured with a microplate reader, and the sample concentration was obtained by converting the A value of the standard product. The results show that CSP-IFN can significantly inhibit the secretion of HBsAg and HBeAg.

[0054] (2) The specific targeting effect of IFN-CSP on liver cells: paraffin-embedded mouse normal liver, kidney, heart, lung, and spleen tissues were taken respectively, and after routine deparaffinizatio...

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Abstract

The invention discloses a fusion protein of hepatic-targeted peptide and human interferon a2b, and a preparation method and application thereof. The nucleotide sequence of the fusion protein of hepatic-targeted peptide and human interferon a2b provided in the invention is described by SEQ ID NO. 1, and the amino acid sequence coded by the nucleotide sequence is described by SEQ ID NO. 2. According to in vitro tests, the fusion protein provided in the invention has obvious activity of resisting HBV and hepatocyte targeting. Since characteristics of both hepatic-targeted peptide of plasmodium circumsporozoite protein CSPI-plus and human IFNa2b are combined into the fusion protein in the invention, the fusion protein can be used for preparation of novel hepatic-targeted antiviral drugs and is of profound significance for lowering down morbidity and mortality of liver diseases caused by infection of HBV. According to the invention, the prepared fusion protein has a high expression level and is easy to purify, production period for the fusion protein is short, and low cost is realized; therefore, the invention has important application prospects and is of practical significance.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein of liver-targeting peptide and human interferon a2b and its preparation method and application. Background technique [0002] Hepatitis B virus (HBV) is a hepatotropic virus. At present, HBV infection has become a global hazard. Among the 6 billion people in the world, about 2 billion people have experienced HBV infection, and 350 million people are chronically infected with HBV , about 80% of patients have varying degrees of liver cell damage, and may develop into liver cirrhosis and liver cancer. More than 1 million people die from hepatitis B-related diseases every year. WHO has listed HBV infection as one of the top ten most common causes of death in the world. my country is a high prevalence area of ​​HBV, and the HBsAg carrier rate of the general population is as high as 7.18%, which not only seriously affects the health of the people, but also is a serious ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N15/70C12N1/15C12N1/19C12N1/21C12N5/10A61K38/21A61K47/48A61P1/16A61P31/20
CPCY02A50/30
Inventor 朱家勇卢雪梅金小宝沈娟马艳梅寒芳褚夫江李小波吴强肖明珠黄演婷
Owner GUANGDONG PHARMA UNIV
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