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Identification and application of pig MHC II TA gene as immunity related molecular labels

A molecular marker and gene technology, which is applied in the determination/inspection of microorganisms, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of few studies on pig MHCIITA gene and no reports of genetic polymorphisms.

Inactive Publication Date: 2009-11-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, there are few studies on the pig MHCIITA gene, and there is no related research report on its genetic polymorphism

Method used

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  • Identification and application of pig MHC II TA gene as immunity related molecular labels
  • Identification and application of pig MHC II TA gene as immunity related molecular labels
  • Identification and application of pig MHC II TA gene as immunity related molecular labels

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Cloning of CDS region of MHCIITA gene

[0028] 1. Primer Design

[0029] The primer design software Primer 5.0 was used to design amplification primers using the porcine MHCIITA gene mRNA sequence on NCBI (Genebank accession number AY084053.1) as a template. The primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the nucleotide sequences of the synthesized primer pairs are as follows:

[0030] Forward primer: 5′-TGGTGACTGGCACTTTCCT-3′,

[0031] Reverse primer: 5'-TGAGCATTGGGTGGGG-3'.

[0032] 2. Purification and sequencing of PCR products

[0033] (1) PCR amplification

[0034] The total volume of the PCR reaction was 10 μL, in which the template DNA was 50 ng, containing 10 × buffer (including Mg2+, Takara), the final concentration of dNTP was 75 μmol / L, the final concentration of primers was 0.3 μmol / L, 1 U Taq DNA polymerase (Takara), The insufficient part was made up with double distilled water.

[0035] The PCR amplification program w...

Embodiment 2

[0066] Detection of the distribution of PCR-PvuII-RFLP polymorphism in pig MHCIITA gene in different breeds

[0067] (1) PCR-PvuII-RFLP detection

[0068] PCR-PvuII-RFLP was used to detect the DNA samples of 6 breeds of pigs, including 3 Chinese local pig breeds, namely Erhualian pig, Dahuabai pig and Tibetan pig; 3 foreign pig bloodlines were Landrace pig and Large White pig and Duroc pigs.

[0069] (2) Analysis of test results

[0070] The genotype individuals and allele frequencies of the mutation C188-T188 in different pig breeds are shown in Table 1. The results showed that allele C was dominant in Chinese local pigs, that is, the 188th base in the 3′UTR region mainly existed in the form of base C; only CT genotype individuals appeared in Tibetan pigs, and the rest were all CC genotypes. The allele C is also dominant in foreign pigs, but the occurrence frequency of T is significantly higher than that of domestic pig breeds; all three genotypes can be detected in foreig...

Embodiment 3

[0077] Application of the pig MHCIITA molecular marker cloned in the present invention in correlation analysis of immune traits

[0078] (1) Construction of family and genotype scanning

[0079]The immune trait resource family used for association analysis is a purebred Landrace pig population (exotic pig blood) jointly established by Huazhong Agricultural University and Guangdong Huanong Wen's Animal Husbandry Co., Ltd. The parents are 17 Landrace boars and 36 Landrace Pig sows, 302 F1 generation Landrace piglets. Anticoagulant and non-anticoagulant blood samples were collected from the F1 generation at 0, 17, and 32 days of age, and conventional methods were used to measure the antibody levels of the three viruses (porcine reproductive and respiratory syndrome virus antibody levels, classical swine fever virus antibody levels, pseudorabies Toxic antibody level) and 18 routine blood tests.

[0080] According to the population structure and influencing factors of the immune ...

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Abstract

The invention belongs to the technical field of livestock molecular label preparation, in particular relates to molecular label preparation and application as auxiliary selection and related to pig immunity property. The molecular label takes mRNA sequence of a pig MHCIITA gene as a template design primer to carry out PCR amplification, PCR product purification and sequencing so as to acquire a nucleic acid shown as a sequence table SEQ ID NO:2; 9 single-chain nucleic acid polymorphic sites (SNP) are found in the nucleic acid sequence shown in the SEQ ID NO:2, and a PCR-PvuII-RFLP method is developed to detect the molecular label on the 188th bp of the nucleic acid sequence, namely base mutation C188-T188. The invention discloses the sequence amplifying a partial code region and a non-translational region of the pig MHCIITA gene, the primer used in PCR amplification and a detection method for the molecular label.

Description

technical field [0001] The invention belongs to the technical field of livestock molecular marker preparation, and in particular relates to the identification and application of a pig MHCIITA gene as an immune-related molecular marker. Background technique [0002] Pork is the main source of animal protein food for Chinese residents, and domestic pigs play a pivotal role in animal husbandry. For decades, the genetic improvement of domestic pigs has achieved remarkable results, but the main focus is on production traits, that is, growth traits and reproductive traits. Compared with production traits, research on pig health and disease resistance is relatively weak. Modern breeding aimed at high-lean pigs has caused the loss or frequency reduction of related alleles, resulting in a sharp decline in the physique and disease resistance of modern commercial breeds. With the popularization of intensive feeding methods, pig diseases, especially some infectious diseases of the res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 赵书红刘向东李新云程文科余梅朱猛进李长春曹建华李世军
Owner HUAZHONG AGRI UNIV
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