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Echinococcus multilocularis glucose transporter polypeptide vaccine GLEP as well as preparation method and application thereof

A technology for glucose transport and multilocular hydatid, which is applied in the application and preparation field of the multilocular hydatid glucose transporter polypeptide vaccine GLEP, can solve the problems of drug resistance, large side effects, and poor treatment effect of human hydatid disease and other problems, to achieve the effect of strong stability, high safety and high purity

Active Publication Date: 2021-06-18
QINGHAI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Drug therapy mainly uses albendazole, praziquantel and oxbendazole, etc., but according to statistics, the cure rate of drugs for echinococcosis is only 30% to 40%, and the therapeutic effect on human echinococcosis is not enough. Good, relatively large side effects, and potential risks such as drug resistance

Method used

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  • Echinococcus multilocularis glucose transporter polypeptide vaccine GLEP as well as preparation method and application thereof
  • Echinococcus multilocularis glucose transporter polypeptide vaccine GLEP as well as preparation method and application thereof
  • Echinococcus multilocularis glucose transporter polypeptide vaccine GLEP as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: Construction of recombinant expression vector pCzn1-GLEP (containing fusion gene GLEP)

[0041] The amino acid sequence of GLEP was converted into the corresponding nucleotide sequence according to the codon preference principle of Escherichia coli, and the full-length splicing primer was designed based on the PAS (PCR-based Accurate Synthesis) method, and protection was designed at both ends of the primer. The sex base synthesis gene GLEP was connected into the expression vector pCzn1 through the cloning sites Nde I and Xba I.

[0042] Results: The recombinant plasmid pCzn1-GLEP to be detected was digested with Nde I and Xba I, reacted at 37°C for 2 hours, and detected by 1% agarose gel electrophoresis. The theoretical size of the same, such as figure 1 shown. The vector construction map of the recombinant expression vector pCzn1-GLEP is as follows: figure 2 shown. The obtained recombinant plasmid pCzn1-GLEP was transformed into the TOP10 clone strai...

Embodiment 2

[0043] Embodiment 2: the prokaryotic expression of polypeptide fusion protein GLEP

[0044] The correct recombinant expression plasmid pCzn1-GLEP was verified to be transferred into Escherichia coli Arctic Express strain. On the pre-prepared LB plate containing 50 μg / mL Amp, inoculate the loop-streaked genetically engineered strain pCzn1-GLEP / ArcticExpress, place it upside down in a 37°C incubator, and after culturing overnight, pick a single colony and inoculate it on a plate containing 50 μg / mL In the LB medium of Amp, cultivate overnight at 37°C, 220rpm. Inoculate the recombinant bacteria with 2% inoculation amount in LB medium containing 50 μg / mL Amp, 37 ° C, 220 rpm, cultivate until the OD600 of the bacteria is 0.6-0.8 (about 2 hours), add IPTG to make the final concentration reach 1 mmol / L, The expression was induced at 37°C and 220rpm for 4 hours, and the carrier strain pCzn1-GLEP / Arctic Express induced without IPTG was used as a negative control.

[0045] Results: Co...

Embodiment 3

[0046] Embodiment 3: Purification of polypeptide fusion protein GLEP

[0047] (1) Cell disruption of recombinant bacteria

[0048] Resuspend the cell pellet in 20ml lysis buffer (20mM Tris-HCl containing 1mM PMSFand bacteria protease inhibitor cocktail, pH 8.0), ultrasonically break (power 400W, work 4sec, intermittent 8sec, total 20min); the ultrasonically broken cell lysate Centrifuge at 10,000 g for 20 min at 4°C and collect the supernatant;

[0049] (2) Purification of Ni-IDA nickel ion affinity chromatography column

[0050] Using the low-pressure chromatography system, the protein solution was loaded onto the Ni-IDA-Sepharose CL-6B affinity chromatography column pre-equilibrated with Ni-IDA Binding-Buffer at a flow rate of 0.5ml / min; Wash at a flow rate of 1mL / min until the OD280 value of the effluent reaches the baseline; wash with Ni-IDA Washing-Buffer (20mM Tris-HCl, 20mM imidazole, 0.15M NaCl, pH8.0) at a flow rate of 1mL / min until the OD280 value of the effluent r...

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Abstract

The invention discloses an echinococcus multilocularis glucose transporter polypeptide vaccine GLEP as well as a preparation method and application thereof. The active ingredient of the vaccine GLEP is a polypeptide, and the polypeptide is mainly composed of an echinococcus multilocularis glucose transporter polypeptide sequence. The echinococcus multilocularis vaccine GLEP gene sequence is synthesized through a gene synthesis technology, double digestion is connected into an expression vector, and then the expression vector is converted into Arctic Express for expression of fusion protein. After the protein is purified through Ni-NTA affinity chromatography, the echinococcus multilocularis vaccine GLEP is obtained. The echinococcus multilocularis vaccine GLEP can induce an organism to generate immune response and high-titer specific antibody humoral immune response for echinococcus multilocularis T cells and B cells, therefore, mice are effectively prevented from being infected with echinococcus multilocularis, and the echinococcus multilocularis glucose transporter polypeptide vaccine GLEP can be used for preventing echinococcus multilocularis infection related diseases.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to the application and preparation method of a glucose transporter polypeptide vaccine of Echinococcus multilocularis GLEP. Background technique [0002] Multilocular echinococcosis is a serious disease with complex clinical symptoms, which may be accompanied by liver pain, shortness of breath, chest pain and other symptoms. Because of its malignant invasive growth, it is called insect cancer. Severe complications occur in the late stage, which endangers the life of the patient. If it is not treated in time, the 10-year mortality rate is as high as 94%, which seriously endangers the physical and mental health of the herdsmen. At present, the main treatment methods include surgery and drug therapy. Surgical treatment is currently the preferred treatment option, but there are serious complications such as high recurrence rate, bile fistula, and abnormal liver function. Drug the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70C12N1/21A61K39/00A61P33/10C12R1/19
CPCC07K14/43536C12N15/70A61K39/0003A61P33/10A61K2039/57
Inventor 李润乐汤锋华国勇格日力冯琳魏威王蕾
Owner QINGHAI UNIVERSITY
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