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Echinococcus multilocularis leucine aminopeptidase subunit vaccine LAP, preparation method and application thereof

A leucine amide peptidase subunit and multilocular hydatid technology is applied in the field of application and preparation of multilocular hydatid subunit vaccine leucine amide peptidase LAP, and can solve the risk of recurrence, poor patient compliance and side effects. It can prevent mice from infecting Echinococcus multilocularis, with high safety and stability.

Pending Publication Date: 2020-08-14
QINGHAI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, benzimidazole (albendazole or mebendazole) is mostly used in the treatment of echinococcosis, but the course of treatment is long, side effects are large, and patient compliance is poor.
According to the survey statistics, although echinococcosis patients can be cured by surgery, there is still a risk of recurrence after surgery. Some patients need to take medicine for a long time or undergo surgery again, which will endanger the physical and mental health of the people and affect the normal operation of animal husbandry.

Method used

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  • Echinococcus multilocularis leucine aminopeptidase subunit vaccine LAP, preparation method and application thereof
  • Echinococcus multilocularis leucine aminopeptidase subunit vaccine LAP, preparation method and application thereof
  • Echinococcus multilocularis leucine aminopeptidase subunit vaccine LAP, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of recombinant expression vector pCzn1-LAP (containing fusion gene LAP)

[0044] The amino acid sequence of LAP was transformed into the corresponding nucleotide sequence according to the principle of E. coli codon preference, and the method based on PAS (PCR-based Accurate Synthesis) was adopted to design the full-length splicing primer, and the protection was designed at both ends of the primer. The sex base synthesis gene LAP is connected to the expression vector pCzn1 through the cloning sites Nde I and Xba I.

[0045] Results: The recombinant plasmid pCzn1-LAP to be tested was double digested with Nde I and Xba I, reacted at 37°C for 2 hours, and detected by 1% agarose gel electrophoresis. It was found that the double digested DNA fragment was about 1700 bp, and the fusion gene LAP The theoretical size of is the same, such as ( figure 1 ) Shown. The vector construction map of the recombinant expression vector pCzn1-LAP is as follows ( figure 2 ...

Embodiment 2

[0046] Example 2: Prokaryotic expression of multi-epitope peptide fusion protein LAP

[0047] The correct recombinant expression plasmid pCzn1-LAP was transformed into the Escherichia coli Arctic Express strain. On the pre-prepared LB plate containing 50μg / mL Amp, inoculate the genetically engineered strain pCzn1-LAP / ArcticExpress, placed upside down in a 37℃ incubator, after overnight culture, pick a single colony and inoculate it with 50μg / mL Cultivate overnight in Amp's LB medium at 37°C and 220 rpm. Inoculate the recombinant bacteria in 50μg / mL Amp LB medium with 2% inoculum, at 37°C, 220rpm, culture until the OD600 of the bacteria is 0.6-0.8 (about 2h), add IPTG to make the final concentration 1mmol / L, The expression was induced at 37°C and 220 rpm for 4 hours, and the carrier strain pCzn1-LAP / Arctic Express without IPTG induction was used as a negative control.

[0048] Results: Compared with the control strain, the genetically engineered recombinant strain pCzn1-LAP / Arctic...

Embodiment 4

[0049] Example 4: Purification of multi-epitope peptide fusion protein LAP

[0050] (1) Denaturation of inclusion body protein

[0051] Resuspend the bacterial pellet in 20ml lysis buffer (20mM Tris-HCl containing 1mM PMSF and bacteria protease inhibitor cocktail, pH 8.0), ultrasonically disrupt (power 400W, work 4sec, interval 8sec, total 20min); ultrasonically disrupt the cell lysate Centrifuge at 10000g for 20min at 4℃, and collect the precipitate; wash the inclusion bodies 3 times with the inclusion body washing solution (20mM Tris, 1mM EDTA, 2M urea, 1M NaCl, 1% Triton X-100, pH8.0); Tris, 5mM DTT, 8M urea (pH8.0), dissolve the inclusion bodies according to a certain proportion, and place overnight at 4℃; centrifuge at 15000rpm for 15min at room temperature; add the above solution dropwise to 20mM Tris-HCl 5mM EDTA Buffer PH7.8, gradually Dilute in multiples with slow stirring, put the protein solution into a dialysis bag and dialyze in PBS pH7.4 solution overnight.

[0052] (...

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Abstract

The invention discloses an echinococcus multilocularis leucine aminopeptidase subunit vaccine LAP, a preparation method and application thereof, and the active ingredient of the subunit vaccine LAP isa polypeptide and is mainly composed of an amino acid sequence of echinococcus multilocularis antigen protein LAP. An echinococcus multilocularis subunit vaccine LAP genetic sequence is synthesized mainly through a gene synthesis technology and is connected to an expression vector through double digestion, and then the expression vector is transformed into Arctic Express for fusion protein expression. After the protein is purified, the echinococcus multilocularis subunit vaccine LAP is obtained. The echinococcus multilocularis subunit vaccine can induce an organism to generate T cell and B cell immune responses and high-titer specific antibody humoral immune responses aiming at echinococcus multilocularis, can effectively prevent mice from infecting echinococcus multilocularis, and can beused for preventing and treating 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 multilocular hydatid subunit vaccine leucine amide peptidase LAP. Background technique [0002] Echinococcus multilocularis infects humans as alveolar echinococcosis (AE), which is a parasitic disease that is zoonotic. From 2012 to 2016, a national survey of echinococcosis in China showed that about 50 million people were at risk of infection in 9 western provinces, and nearly 170,000 people were infected with echinococcosis. Most of the primary lesions of Echinococcus multilocularis are in the liver, and most patients with hydatid disease go to a doctor late. Early hydatid disease does not cause symptoms, and AE lesions can remain asymptomatic for 10 to 15 years. Clinical symptoms usually appear when the diameter of the intrahepatic cyst exceeds 10 cm, or more than 70% of the organ volume is occupied by the cyst or cyst, resulting in ...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61P33/10C12N15/70C12N1/21C12N15/62C12R1/19
CPCA61K39/0003A61P33/10C07K14/43504C07K2319/00
Inventor 嘎琴李润乐樊海宁格日力汤锋杨宝良郑佳冯琳王蕾胡缤文
Owner QINGHAI UNIVERSITY
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