Method for obtaining wire-worm microsome amino peptidase gene and use thereof

A technology of twisting Haemonchus contortus and microsomes, which is applied in the biological field to achieve high stability, biological activity and complete sequence

Inactive Publication Date: 2009-05-20
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] How to improve the immune protection of H11 recombinant form has become the focus of many scholars. Mastering the activity of H11 promoter and understanding the regulation rules

Method used

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  • Method for obtaining wire-worm microsome amino peptidase gene and use thereof
  • Method for obtaining wire-worm microsome amino peptidase gene and use thereof
  • Method for obtaining wire-worm microsome amino peptidase gene and use thereof

Examples

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Embodiment 1

[0034] This example describes the method for obtaining the cDNA sequence of the H11 gene of H. contortus Zhejiang strain provided by the present invention, and the whole test process of long-distance RT-PCR amplification and cloning of the H11 cDNA.

[0035](1) RT-PCR amplification of the cDNA sequence of H. contortus Zhejiang strain H11

[0036] 1. Refer to the third edition of Molecular Cloning to prepare RNA from tissues with one-step method and extract H. contortus total RNA. After formaldehyde gel electrophoresis confirms that there is no degradation, store at -80°C.

[0037] 2. Primer design: according to the sequence published by Smith (1997), according to the sequence characteristics of its reading frame, the two ends were respectively added with Sal I and HindIII restriction sites and protected bases to design primers P1 / P2 (refer to SEQ NO 1 and SEQ NO 2), the primers were synthesized by Shanghai Boya Biotechnology Co., Ltd.

[0038] 3. Synthesis of cDNA of H. conto...

Embodiment 2

[0046] This example describes the process of obtaining the genome sequence of the H11 gene mainly by using the gene walking technology in the present invention.

[0047] (1) Acquisition of H11 partial genome sequence: Take 50 mg of H. contortus washed with PBS buffer and transfer it to a Collection Tube in an ice bath, quickly grind it into a paste with a grinding rod, and extract it according to the method of TaKaRa Universal Genomic DNA Extraction Kit Genomic DNA, stored at -20°C.

[0048] Using the extracted H. contortus genomic DNA sequence as a template, a long-distance PCR reaction was performed using primers Hc-GSF / Hc-GSR (refer to SEQ NO 5 and SEQ NO 6). Reaction system (50μl): TaKaRa LA Taq (5U / μl) 0.5μl, 10×LA PCR Buffer (Mg 2+ Plus) 5 μl, dNTPMixture (2.5 mM each) 8 μl, genomic DNA 0.7 μl, Hc-GSF (20 μM) 1 μl, Hc-GSR (20 μM) 1 μl, ddH 2 O 38 μl. The reaction conditions were pre-denaturation at 94°C for 3min; 94°C for 60s, 58°C for 60s, 72°C for 6min; 72°C for 10m...

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Abstract

The invention provides a method for obtaining a haemonchus contortus (H.contortus) microsomal aminopeptidase gene which comprises the steps of total RNA extraction, H11 cDNA sequence clone, homology comparison and amino acid sequence analysis of H11 cDNA sequence as well as H11 genome sequence clone and structural analysis. By referring to the H11 gene cDNA sequence, the method can conveniently, completely and accurately obtain H11 genome sequence, can analyze the H11 mRNA transcription characteristic by analyzing the H11 genome sequence, and provides reliable background material and data for developing H.contortus vaccines. The haemonchus contortus (H.contortus) microsomal aminopeptidase gene that is provided by the invention can be applied to the subunit vaccine preparation that uses recombinant proteins.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to a method for obtaining the microsomal aminopeptidase gene of Haemonchus contortus Zhejiang strain and its application. Background technique [0002] Haemonchus contortus (Haemonchus contortus) is the main parasitic nematode of ruminants such as cattle and sheep, and the haemonchus contortus disease caused by it causes millions of economic losses to my country's agricultural production every year. Drug control is the main method to control the disease, but the increasing drug resistance of nematodes and the emergence of drug residues restrict the long-term use of existing anti-nematode drugs. As a new control strategy, vaccine development has become a research hotspot. [0003] Microsomal aminopeptidase H11 is an integral membrane glycoprotein complex with a molecular weight of 110 kDa expressed only on the microvilli of the small intestine during the parasitic stage. H11 be...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N9/48A61K39/00A61P33/10
Inventor 杜爱芳周前进张红丽姜小磊
Owner ZHEJIANG UNIV
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