Recombinant porcine beta 2-adrenergic receptor protein and application thereof

A technology of adrenaline and recombinant protein, applied in the field of bioengineering, can solve the problems of poor activity and achieve the effect of ensuring activity and accuracy

Inactive Publication Date: 2014-08-13
INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Domestic for β 2 Most of the research on AR has focused on the cloning and expression of the receptor gene. The expressed receptor protein has either not been identified for activity, or the activity has been proved to be poor by experiments, and the recombinant receptor has been applied to β 2 Research on agonist detection is almost blank

Method used

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  • Recombinant porcine beta 2-adrenergic receptor protein and application thereof
  • Recombinant porcine beta 2-adrenergic receptor protein and application thereof
  • Recombinant porcine beta 2-adrenergic receptor protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 β 2 Adrenergic receptor gene Ss β 2 PCR amplification of AR

[0035] Beta of porcine (Sus scrofa) retrieved on NCBI 2 AR sequence (Genbank accession number AF000134), according to its two-terminal sequence, design a pair of primers, the 5' end of the upstream primer is introduced into the Nco Ⅰ restriction site, and the 5' end of the downstream primer is introduced into the Xhol Ⅰ restriction site, by RT-PCR β was amplified from the isolated and purified porcine hepatocyte total RNA 2 Adrenergic receptor gene Ss β 2 AR, its gene sequence is shown in SEQ ID No.2; its coded amino acid sequence is shown in SEQ ID No.1. The upstream primer (SEQ ID No.3) is: 5'-CAT GCCATGG CAGGGCAGCCCGGGAACCGC-3', the underlined sequence is the Nco I restriction site. The downstream primer (SEQ ID No.4) is 5'-CCG CTCGAG TCACAGCATGGAGTCATTTGTACTACAGTTCCTCC-3'. The underlined sequence is the Xhol I restriction site.

[0036] The reaction system is as follows:

[0037] ...

Embodiment 2

[0039] Example 2 β 2 Adrenergic receptor gene Ss β 2 Sequence analysis of AR and its encoded protein

[0040] beta 2 Adrenergic receptor gene Ss β 2 AR and published pig β 2 Compared with the AR gene (AF000134), the gene sequence identity is 99.68%, four base mutations occur, the amino acid sequence identity is 99.28%, three mutations occur, and β 2 Amino acids at the binding site of the agonist and the receptor are cloned correctly, which is suitable for the expression of active β in vitro 2 AR laid the groundwork.

Embodiment 3

[0041] Embodiment 3PMD-18T-Ss β 2 Construction and Identification of AR Recombinant Cloning Plasmid

[0042] 1) PCR product purification

[0043] After the PCR product was subjected to 1% agarose gel electrophoresis, the target fragment was recovered by cutting the gel according to the operation steps of the DNA recovery and purification kit.

[0044] 2) Connection of target gene and cloning vector

[0045] The recovered product and the PMD-18T vector were double-digested with Nco Ⅰ and Xhol Ⅰ, respectively, and after 1% agarose gel electrophoresis, the double-digested product of the target gene and the large plasmid fragment were recovered. According to the molar ratio of 3:1, the T4 ligase Under the action, connect overnight at 4°C.

[0046] 3) Transformation and identification of recombinant cloning plasmids

[0047] The ligation product was transformed into DH5α competent cells and plated, and a single white colony was picked to extract the plasmid for PCR identificati...

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Abstract

The invention belongs to the field of bio-engineering, and particularly relates to a method for preparing recombinant porcine beta 2-adrenergic receptor protein and an application of the recombinant porcine beta 2-adrenergic receptor protein in rapidly detecting beta 2 agonist medicaments. The recombinant porcine beta 2-adrenergic receptor protein obtained by separation and purification has beta 2 agonist binding activity, SDS-PAGE electrophoresis shows that the molecular weight of the recombinant protein is about 48kDa and the purity of the recombinant protein is more than 80%. The recombinant protein can specifically adsorb beta 2 agonist medicaments and three beta 2 agonist medicaments standard products with different concentrations have an inhibition effect on the reaction when detection is carried out by a competitive ELISA method. According to method disclosed by the invention, the beta 2AR receptor capable of specifically binding with beta 2 agonist is efficiently expressed by a molecular biology method and can be used for rapidly screening beta 2 agonist medicaments instead of conventional antibodies to achieve the detection of multi-residue and screening of unknown substance in medicaments, and the method has good application prospects.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a recombinant pig β 2 Preparation method of adrenergic receptor protein, and its expression in β 2 Application in rapid detection of agonist drugs. Background technique [0002] my country has banned the use of beta in animal feed and drinking water 2 agonist, but its illegal abuse still occurs from time to time, seriously affecting the quality and safety of animal products and people's health. Therefore, strengthening the β 2 The detection and regulation of agonist drugs has important practical significance. [0003] Currently, for β 2 There are mainly the following types of agonist residue detection techniques. (1) Chromatographic detection method. Commonly used chromatographic techniques are high-performance liquid chromatography (HPLC) (Degroodt et al., 1989), gas chromatography-mass spectrometry (GC-MS) (Fente et al., 1999), high-performance liquid chromatography-mass sp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/72C12N15/12C12N15/85C12N5/10G01N33/68
CPCC07K14/723C12N15/85G01N33/68G01N33/94G01N2500/04
Inventor 王静王健王淼金茂俊佘永新杜欣蔚刘媛金芬邵华王珊珊
Owner INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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