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An apyrase encoding gene of Aedes albopictus, and preparation method and application of protein thereof

Aedes albopictus and protein technology, applied in botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the difference in titer and specific antigen level, it is difficult to standardize and quantify, and limit the application of diagnosis and treatment, etc. problems, to achieve high output and constant production conditions

Inactive Publication Date: 2013-01-02
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the traditional mosquito body leachate is used in the clinical diagnosis of patients with mosquito allergy, which contains a variety of antigenic components, and there are obvious differences in relative potency and specific antigen level, it is difficult to standardize and quantify, and it is easy to cause patients to have a diagnosis Hypersensitivity reactions, thus limiting its application in diagnosis and treatment

Method used

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  • An apyrase encoding gene of Aedes albopictus, and preparation method and application of protein thereof
  • An apyrase encoding gene of Aedes albopictus, and preparation method and application of protein thereof
  • An apyrase encoding gene of Aedes albopictus, and preparation method and application of protein thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Preparation of Aedes albopictus salivary gland

[0037] Adult (1-5 days old) non-blood-sucking Aedes albopictus, select female mosquitoes, remove wings, pick salivary glands under a dissecting microscope, wash with PBS, and store at -80°C for later use.

Embodiment 2

[0038] Example 2 Acquisition of Aedes albopictus Apyrase Encoding Gene

[0039] Using the RNeasy Plus minikit, according to the instructions of the kit, total RNA of Aedes albopictus was extracted and stored at -70°C for future use. RT-PCR was performed using ProtoScriptTM First Strand cDNA Synthesis kit to obtain cDNA. Using APY-Nde I-S 5'-CCCATATGATGGATAATATGCCCGCTGATAAAGATG-3', APY-XhoI-AS 5'-CCCTCGAGTCAAGTACATGGTGAACCTGCTTTACAT-3' as template, PCR amplified the gene fragment encoding Apyrase, and the PCR product was detected by 0.8% agarose gel electrophoresis. Its size is 1620bp (such as figure 1 shown).

Embodiment 3

[0040] Example 3 Construction of recombinant expression plasmid pET19b-Apyrase

[0041] The PCR amplification product was purified with QIAquick PCR Purification Kit, and then the PCR amplification product was digested with NdeI and XhoI and inserted into the pET19b vector. Transform into Escherichia coli JM109, spread the bacterial solution on LB agar plates (containing ampicillin 100 μg / ml, chloramphenicol 34 μg / ml), culture overnight at 37°C, pick clones, extract recombinant plasmids for double enzyme digestion identification , the results showed that the target band was consistent with the size of the PCR product of the Apyrase gene fragment, indicating that the recombinant plasmid pET19b-Apyrase of Aedes albopictus Apyrase (such as figure 2Shown) The recombinant plasmid was sequenced and identified by Shanghai Handsome Biotechnology Co., Ltd., and the amino acid sequence was deduced using GENETYX-MAC Ver.11.0 software, and compared with AAC37218.1 (Aedes aegypti apyrase)...

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Abstract

The invention falls into the field of biotechnology, and relates to an apyrase encoding gene of Aedes albopictus, and preparation method and application of protein thereof. In the invention, RNA is extracted from the salivary gland of Aedes albopictus; the apyrase encoding gene of Aedes albopictus is obtained by PCR; and an apyrase expression vector pET19b-apyrase is constructed. After abundant expression, purification and further identification, it has been proved that, the recombinant apyrase of Aedes albopictus has equal immune activity as natural apyrase of Aedes albopictus, can cause immune response of human, and has inhibitory activity against platelet aggregation. The inventive apyrase of Aedes albopictus can be used in the diagnosis of anaphylactic diseases induced by Aedes albopictus, and also be used as antithrombotic for treating thrombus caused by platelet aggregation.

Description

technical field [0001] The invention relates to the field of biotechnology, and relates to an Apyrase coding gene of Aedes albopictus and a protein preparation method and application thereof. Background technique [0002] Literature discloses that Apyrase is a hydrolase that can hydrolyze nucleotide triphosphate (ATP) and nucleotide diphosphate (ADP) into monophosphate nucleotide and inorganic phosphorus. Apyrase is found in plants, arthropods and vertebrates. It has been reported that many seemingly unrelated proteins such as heat shock protein, actin and glycokinase also have the activity of hydrolyzing ATP and ADP. Valenzuela believes that these proteins are the products of convergent evolution. In general, nucleotidases can be divided into three categories based on amino acid sequence and substrate specificity: exogenous, 5'-nucleotidases derived from the CD73 lymphocyte protein family, and nucleotidases derived from blood-sucking arthropods. Nucleic acid hydrolase. T...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/55C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12Q1/44A61K38/46A61P7/02
Inventor 程训佳付永锋董芳周承俊
Owner FUDAN UNIV
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