HPSE (Heparanase) gene as molecular markers relevant to pig immune characters and application thereof

A molecular marker and molecular marker-assisted technology, which is applied in the field of molecular marker-assisted selection in pigs, can solve problems such as disease prevention that cannot be fundamentally solved.

Inactive Publication Date: 2010-11-24
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But none of them can fundamentally so

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  • HPSE (Heparanase) gene as molecular markers relevant to pig immune characters and application thereof
  • HPSE (Heparanase) gene as molecular markers relevant to pig immune characters and application thereof
  • HPSE (Heparanase) gene as molecular markers relevant to pig immune characters and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1. Cloning of HPSE gene

[0023] (1) Primer design

[0024] Using human HPSE gene cDNA (GenBank accession number: NM_006665.3) as an information probe, using the BLAST tool in NCBI to screen homologous sequences in the GenBank pig EST database, a series of ESTs (fragments) with a homology of more than 80% were obtained. Length greater than 100bp), use ENTREZ (http: / / www.ncbi.nlm.nih.gov / Web / Search / index.html) to query the corresponding sequence in NCBI with the indexing number of these ESTs, and then use the ASSEMBLY program in GeneTool Construct a pig EST-contig. Design a pair of primers M-F and M-R according to the EST splicing sequence, the sequence is as follows

[0025] HPSE: M-F: 5'-AGCCAGGTGAGCCCGAGATG-3',

[0026] M-R: 5'-GCATCTGCTCGTGTTCCTAC-3'.

[0027] (2) Cloning and sequencing of PCR products

[0028] Connect the purified PCR product to the pMD-18T vector (purchased from Takara) in a water bath at 4°C overnight; take 100-120μl of competent cells into a 1.5m...

Embodiment 2

[0031] Example 2. Establishment of PCR-RFLP diagnostic method

[0032] (1), primer sequence

[0033] HPSE P-SF 5'-GGATGAAGGCTGGTATTT-3' (corresponding to SEQ ID NO: 3)

[0034] P-SR 5'-TGGGATAAGGCAATACAG-3' (corresponding to SEQ ID NO: 4)

[0035] (2) PCR amplification conditions

[0036] The total volume of PCR reaction is 20μl, of which porcine genomic DNA is about 100ng, containing 1 times buffer (purchased from Promega), 1.5mmol / LMgCl 2 , The final concentration of dNTP is 150μmol / L, the final concentration of primer is 0.4μmol / L, 2U Taq DNA polymerase (Promega). The PCR amplification program is: 94°C for 3min, 35 cycles of 94°C for 30s, 54°C for 30s, then 72°C for 25s, and finally 72°C for 5min. PCR reaction products were detected by 2% agarose gel electrophoresis. A 374bp specific amplified fragment is obtained, which is located in the 4-5th intron (such as figure 2 ). The result of sequencing revealed that there is an Alu I restriction site (AG↓CT) in the 374bp fragment, and ...

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Abstract

The invention belongs to the technical field of the preparation of livestock molecular markers and particularly relates to preparation and application of pig molecular markers. The preparation method comprises the steps: cloning from a human source HPSE (Heparanase) gene to obtain a gene fragment applied as a molecular marker relevant to pig immune characters, wherein the nucleotide sequence of the gene fragment is disclosed as SEQ ID No:2, and A80-G80 base substitution exists in the 80bp position of the SEQ ID No:2 to lead to the polymorphism of Alu I-RFLP (Restricted Fragment Length Polymorphisms). The invention also discloses a method for preparing molecular markers relevant to pig immune characters and application of the prepared molecular markers to the detection of the polymorphism of the pig immune characters. The invention provides a new marker for the assisted selection of pig markers.

Description

Technical field [0001] The invention belongs to the technical field of pig molecular marker assisted selection, and specifically relates to the cloning and application of a molecular marker related to immune traits as a pig marker assisted selection. The molecular marker of the present invention is related to the HPSE gene. Background technique [0002] In the past 10 years, modern breeding technology has significantly improved the performance of pigs (growth, carcass and meat quality), but insufficient attention has been paid to genetic improvement of pig health and disease resistance. Disease has always been an important link in animal husbandry production. Preventive measures such as improving the management level, improving the sanitation environment, injecting drugs and vaccines, have indeed played an important role in preventing the occurrence of diseases. But none of them can fundamentally solve the problem of disease prevention. Improving the body's natural disease res...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12Q1/68
Inventor 刘榜王维民韩雪蕾杨华威彭中镇王振华李文献
Owner HUAZHONG AGRI UNIV
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