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Garrupa MHC (Major Histocompatibility Complex) IIB gene as well as cloning method and application thereof

A grouper and gene technology, applied in the field of genetic engineering, achieves the effect of high breeding efficiency and simple operation

Inactive Publication Date: 2011-04-13
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research on the MHC gene of grouper at home and abroad.

Method used

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  • Garrupa MHC (Major Histocompatibility Complex) IIB gene as well as cloning method and application thereof
  • Garrupa MHC (Major Histocompatibility Complex) IIB gene as well as cloning method and application thereof
  • Garrupa MHC (Major Histocompatibility Complex) IIB gene as well as cloning method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Extraction of total RNA from grouper spleen

[0032]Healthy 2-year-old grouper (Epinephelus coioides), weighing about 750 g, was anesthetized with MS-222 (tricaine methanesulphonate) for about 1 min, and the spleen was isolated immediately after blood was collected from the tail vein. The total RNA of grouper spleen was extracted by Trizol reagent, and its OD260 / 280=1.85. The electrophoresis results showed that 28S rRNA and 18S rRNA bands were clear, and the brightness of 28S band was twice that of 18S, indicating that the total RNA obtained was not contaminated by protein , phenol and genomic DNA pollution, high purity.

[0033] 2. Synthesis of the first strand of cDNA

[0034] Take 5 μg of the total RNA sample of the grouper spleen and carry out DNase treatment to remove the contamination of genomic DNA, mix with RNA Oligo dT (sequence shown in SEQ ID NO: 33), carry out reverse transcription, and place the product in- Store at 20°C for later use.

[0035] 3. Pri...

Embodiment 2

[0045] Example 2 Sequence polymorphism of the MHC IIB gene of the grouper

[0046] According to the synthesis of specific upstream primer EpcoMHC2bPl-F on the first exon of the grouper MHC IIB gene, the sequence is shown in SEQ ID NO: 31, and the specificity is synthesized at the end of the second exon and the front end of the second intron. The downstream primer EpcoMHC2bPl-R, the sequence is shown in SEQ ID NO: 32; PCR amplification was carried out in 45 different grouper individuals, 145 positive clones were picked and sent for sequencing, and the polymorphism in the PBR region of the MHC IIB gene was obtained Sexual raw data. Sequencing results showed that there were two different sites of MHC IIB gene in grouper, and the difference in molecular weight was due to the length of Intron 1. The size of Intron1 at one site is 138bp, which is named DAB2; the size of Intron1 at another site varies greatly in different fish species, but is always greater than 138bp, and is named ...

Embodiment 3

[0047] Example 3 Screening of MHC IIB gene disease resistance-related markers of the grouper

[0048] Use natural selection and artificial infection to obtain disease-resistant populations and non-disease-resistant populations. In the peak season of the disease, the surviving individuals are collected from the farm, and the collected fry are infected with pathogenic bacteria at the same time, and the disease-resistant population and non-disease-resistant population are obtained according to the resistance and survival ability of the pathogen infection. A total of 321 positive clones from 81 individuals were sequenced, and the results showed that there were 6 MHC genotypes with high frequency in disease-resistant individuals, among which EpcoDAB*1-01, EpcoDAB*1-02, EpcoDAB*1-04 , EpcoDAB*1-05, EpcoDAB*2-01 and EpcoDAB*2-02, the frequencies of occurrence are 14.81%, 7.41%, 7.41%, 14.81%, 7.41%, 11.11% and 37.04% ( figure 1 ).

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Abstract

The invention discloses a garrupa MHC (Major Histocompatibility Complex) IIB gene as well as a cloning method and application thereof. The nucleotide sequence of the garrupa MHC IIB gene is shown as SEQ ID NO:1, and the amino acid sequence of protein encoded by the gene is shown as SEQ ID NO:2. In the invention, by using a homologous cloning method, the MHC IIB gene is separated from garrupa, 12 different MHC IIB gene types are identified, the correlation between the polymorphism and the disease resistance of the MHC IIB gene are analyzed, and 6 molecular marks of the MHC IIB gene relevant to the disease resistance of the garrupa are screened; and the cloning method is suitable for screening the marks relevant to the disease resistance of the garrupa and breeding disease-resistant species.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a grouper MHC IIB gene and its cloning method and application. Background technique [0002] The major histocompatibility complex (MHC) is a highly polymorphic gene group encoding immunoglobulin-like receptors commonly found in vertebrates, which plays an important role in the immune system. It is one of the hotspots in immune research in recent years. MHC genes are divided into types I, II, and III, and type II genes are further divided into IIA and IIB. The products encoded by MHC class II genes are MHC class II molecules, which combine with exogenous antigenic peptides and present them to T helper cells, causing downstream immune responses. Many studies have shown that polymorphisms of MHC II genes are closely related to individual disease resistance. [0003] The MHC class II molecule is a heterodimer consisting of an α-polypeptide chain and a β-polypeptide chain. Both ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/10C07K14/74C12Q1/68
Inventor 张勇易诗白王磊张海发卢丹琪李水生蒙子宁刘晓春林浩然
Owner SUN YAT SEN UNIV
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