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Detection method of pig TLR4 gene T611A base mutation
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Al technical title is built by PatSnap Al team. It summarizes the technical point description of the patent document.
A T611A, detection method technology, applied in the field of genetic engineering, can solve the problems of weakened stimulus response, reduced virus resistance, etc.
Inactive Publication Date: 2013-01-30
JIANGSU ACAD OF AGRI SCI
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[0003] TLR4 plays an important role in the transmembrane signal transduction of pathogenic microorganisms. Studies have confirmed that TLR4 is the main receptor of the body's immune response to LPS. The mice knocked out of this gene have a significantly weakened response to bacterial pathogenic stimulation, and TLR4 gene-deficient mice are resistant to Reduced resistance to the virus
[0004] At present, there are occasional reports on SNP detection of porcine TLR4 gene, but it is basically limited to single base mutations in individual exons
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Embodiment 1
[0032] Acquisition of porcine TLR4 gene cDNA and primer design
[0033] Extract the total RNA of pigs (Landrace pigs, purchased from Jiangsu Changzhou Kangle Farming and Animal Husbandry Co., Ltd.) with TrizolReagent (Invitrogen), and detect the nucleic acid concentration and purity with a UV spectrophotometer; M-MLV reverse transcriptase (Promega) and Oligo ( dT)18 (Promega) reverse-transcribed cDNA; according to the GenBankdatabase porcine TLR4 gene sequence (AB188301, AJ628065), specific primers were designed with Primer 5.0 for PCR amplification. The primer sequences are:
[0034] Sense primer 5'-TTTCTAACCTGCCCAACCTG-3' (SEQ ID NO: 1)
[0035] Antisense primer 5'-GAACCAGCCAGACCTTGAAT-3' (SEQ ID NO: 2)
Embodiment 2
[0037] PCR amplification and product purification
[0038] Using primers SEQ ID NO: 1-2 to carry out PCR amplification of the porcine template cDNA to be detected.
[0039] PCR amplification reaction system (12μl): 10×PCR buffer 1.2μl, 2mM / L dNTPs 1.2μl, 25mM / L MgCl 2 0.8μl, 10pmol / μl primer 1.0μl, 5U / μl Taq polymerase 0.2μl, 50ng / μl cDNA 0.8μl, ddH 2 O6.8μl;
[0040] Reaction conditions: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 1 minute, 34 cycles; extension at 72°C for 8 minutes;
[0041] Wherein, the PCR amplification fragment length is 247bp ( figure 1 ). Use the nucleic acid purification kit to separate and purify the PCR amplification products according to the protocol provided by the manufacturer.
Embodiment 3
[0043] Sequencing and SNP detection
[0044] The purified porcine TLR4 gene SEQ ID NO: 1-2 primer PCR amplification product is directly sequenced, and the sequence is determined by comparing the PCR products of different samples. As a result, in the tested 10 Landrace pig experimental samples, the 611th site of the TLR4 gene cDNA sequence showed 6 Ts and 4 As, revealing that the experimental pigs had a T / A single nucleus at the 611th site of the TLR4 gene coding region. nucleotide base mutation ( figure 1 , 2 ).
[0045] Moreover, the T611A mutation at position 611 of the pig TLR4 gene directly leads to a Leu204His change in the encoded amino acid, and the amino acid properties change accordingly, resulting in a change in the superhelical structure of the functional domain of the TLR4 gene, which affects the ability of TLR4 as a pattern recognitionreceptor to specific pathogens. Immune response, which in turn manifests different immunity to specific diseases. Provide the ...
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Abstract
The invention relates to a detection method of pig TLR4 gene T611A base mutation. The invention belongs to the technical field of genetic engineering. According to the invention, specific primers are designed according to a pig TLR4 gene sequence in the GenBank data base; the primers are used in PCR amplification upon pig template cDNA requiring detection; an amplification product is separated and purified, such that a specific nucleic acid is obtained; the separated and purified amplification product is sequenced; after comparison and analysis, it is determined whether T611A mononucleotide base mutation exists in the amplified sequence. The method provided by the invention can be used in the detection of pig TLR4 gene mononucleotide base mutation. The method has a great significance in the determination of the changes of body immunological recognition, provides a basis of pig plague controlling and breeding for disease resistance, and provides genetic engineering technical assistance for the researches of pig breeding for disease resistance.
Description
(1) Technical field [0001] The invention relates to a detection method for T611A base mutation of porcine TLR4 gene, which belongs to the technical field of genetic engineering. With this method, the single nucleotide base mutation of the porcine TLR4 gene can be quickly detected, which provides assistance for the molecular structure and function research of the porcine immune defensesystemreceptor gene. (2) Background technology [0002] Toll-like receptors (TLRs), as important pattern recognition receptors, mediate the body's natural immunity and acquired immunity through the recognition of pathogen-associated molecular patterns (PAMPs) and signalcascade reactions, and are the first step for the body to resist infection. Tract barrier; TLRs gene mutations lead to loss of the host's ability to respond to Gram-positive and Gram-negative bacteria, changing the body's resistance or susceptibility to pathogens. [0003] TLR4 plays an important role in the transmembrane sign...
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