Yeast expression system for expressing HAS-Vmip-II fusion protein and construction method thereof

An expression system and fusion protein technology, applied in the field of bioengineering, can solve problems such as toxic side effects, increase patient pain and treatment costs, blood drug concentration cannot inhibit virus replication, etc., and achieve the effect of simplifying the screening process

Inactive Publication Date: 2011-10-19
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Taking interferon beta as an example, the half-life of intramuscular injection is generally about 2h-4h. Even after six half-lives (24h) of high-dose administration, its blood concentration can no longer achieve the purpose of inhibiting virus replication.
In order to achieve the best therapeutic effect, frequent high-dose medication is required. Long-term frequent injections not only increase the patient's pain and treatment costs, but also easily cause a series of serious side effects

Method used

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  • Yeast expression system for expressing HAS-Vmip-II fusion protein and construction method thereof
  • Yeast expression system for expressing HAS-Vmip-II fusion protein and construction method thereof
  • Yeast expression system for expressing HAS-Vmip-II fusion protein and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Construction of HAS-vMIP-II Yeast Expression System

[0039] 1. Alkaline lysis method to extract the plasmid containing the target gene from Escherichia coli

[0040] (1) Cultivate Escherichia coli DH5a transformed with the recombinant plasmid pPICZaA-HSA-vMIP-II, and harvest the bacteria: Pour 1.5ml of bacterial liquid into a microcentrifuge tube, centrifuge at 12000g for 30 seconds, and absorb the culture medium. The supernatant must be aspirated clean, otherwise it will affect the quality of the plasmid. Centrifuge twice if necessary.

[0041] (2) Resuspend the bacterial pellet in 100 μl ice-cooled solution I (solution I preparation: 50 mM glucose / 25 mM Tris-HCl / 10 mM EDTA, pH 8.0; store at 4°C after autoclaving) , add 1 / 50 volume of RNase A stock solution, shake vigorously to make it completely dispersed. Glucose can be replaced by NaCl to facilitate storage; to extract plasmids larger than 15kb, the bacteria should be suspended in isotonic sucrose solu...

Embodiment 3

[0102] Example 3 Detection of biological activity of purified protein (chemotaxis inhibition experiment)

[0103] Preparation of PBMC: Take about 4ml of lymphocyte separation solution in a clean centrifuge tube, dilute 4ml of fresh healthy peripheral blood with an equal volume of RPMI1640 solution, carefully add to the lymphocyte separation solution, room temperature 1300 rpm, 12 min ;The solution will be divided into 3 layers, the middle layer of flocculent is the peripheral blood mononuclear cells, take the lymphocyte layer into another clean centrifuge tube, add 4~5ml RPMI1640 solution to wash, 1200 rpm, 8 min; Clear, re-suspend with 4ml RPMI1640 solution (containing 10% calf serum), and calculate the cell density on a cell counting plate.

[0104] Calculate the amount of diluent according to the adjusted cell density to adjust the cell density to 1×10 6 / ml, and resuspended in RPMI1640 medium (containing 10% calf serum).

[0105] The above cell suspension was treated wit...

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Abstract

The invention discloses an expression system for expressing an HAS-Vmip-II fusion protein, and the expression system is produced by the transformation of plasmid pPICZaA-HSA-vMIP-II into Pichia pastoris. The invention also discloses a construction method of the expression system, comprising the following steps of: extracting recombinant plasmid from escherichia coli containing the plasmid pPICZaA-HSA-vMIP-II, linearizing the recombinant plasmid, transforming Pichia pastoris competent cells by electrotransformation, followed by resistance screening, performing the PCR identification to obtain positive clones, carrying out an inducible expression by the use of a positive clone, followed by the SDS-PAGE analysis of the expression product and Western-Blot identification. According to the invention, the half life of vMIP-II in blood plasma is greatly prolonged without the loss of vMIP-II functions. Therefore, the HAS-vMIP-II fusion protein will reduce the administration frequency and dosage, and exert a drug effect similar to that of vMIP-II.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a yeast expression system capable of efficiently expressing HSA-vMIP-II fusion protein and a construction method thereof. Background technique [0002] Kaposi's sarcoma (Kaposi's sarcoma, KS) is a vascular multiple tumor, commonly seen in patients with immunodeficiency diseases. Since the prevalence of AIDS (Acquired Immune Deficiency Syndrome), KS has become one of the most common tumors in Africa. Biopsies of individuals at high risk for KS or possible KS revealed genomic DNA of Kaposi's sarcoma-associate herpsvirus (KSHV), also known as human herpesvirus 8 (HHV8), suggesting the involvement of KSHV in KS pathological process. [0003] Kaposi's sarcoma virus is the causative agent of Kaposi's sarcoma that often occurs in HIV-infected or transplanted patients after receiving immunosuppressive agents. This virus has a total of more than 80 genes, and its K6 and K4 genes encode a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C07K19/00A61K38/38A61K38/19A61K47/48A61P37/02A61P31/22C12R1/84
CPCY02A50/30
Inventor 孙晗笑贾忠伟肖威莫雪梅李秀英张光王峰
Owner JINAN UNIVERSITY
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