Encoding gene from anopheles sinensis aquaporin 2 and application of anopheles sinensis aquaporin 2

An aquaporin and encoding gene technology, applied to the encoding gene and application of Anopheles sinensis aquaporin 2 (AsAQP2), aquaporin and its encoding gene and application field, can solve research problems and achieve efficient water transport Function, the effect of good application prospects

Inactive Publication Date: 2014-04-16
JIANGSU INST OF PARASITIC DISEASES
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Encoding gene from anopheles sinensis aquaporin 2 and application of anopheles sinensis aquaporin 2
  • Encoding gene from anopheles sinensis aquaporin 2 and application of anopheles sinensis aquaporin 2
  • Encoding gene from anopheles sinensis aquaporin 2 and application of anopheles sinensis aquaporin 2

Examples

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Embodiment 1

[0022] Example 1 Cloning of Anopheles sinensis aquaporin 2 cDNA sequence

[0023] According to the conserved amino acid sequence of aquaporin in Anopheles gambiae, the demerger primers were designed, and the full-length cDNA of this gene was cloned from Anopheles sinensis by RT-PCR combined with RACE technology. After sequence comparison, the gene has a high homology with one of the aquaporins in Anopheles gambiae. The expression abundance of this protein in Anopheles gambiae is high, and it has a very efficient water transport function. Design specific primers based on sequence information:

[0024] 3F: 5'-ATGACTGAAA GCGCAGGAGT G-3',

[0025] 3R: 5'-TTAAAAATCG TATGAATTTG ATTCTTCG-3',

[0026] Using the adult Anopheles sinensis mRNA, the cDNA template was obtained using Invitrogen’s SuperScriptⅢ reverse transcription kit, and the PCR reaction was prepared according to the following reaction system:

[0027] cDNA template 2 μL Upstream primer 3F (20 μM) 0....

Embodiment 2

[0031] Example 2 In vitro function identification of Anopheles sinensis aquaporin 2 (Xenopus oocyte system)

[0032] Primers were designed according to the coding region of the gene sequence, and BglⅡ restriction sites were added at both ends. The specific primer sequences are as follows:

[0033] AQP2BglⅡF0': 5'-AGA agatct ATGACTGAAA GCGCAGGAGT G-3',

[0034] AQP2BglⅡR0': 5'-AGA agatctTTAAAATCGT ATGAATTTGA TTC-3',

[0035] The endonuclease BglⅡ digested and recovered the PCR product and the oocyte expression vector pXβG-myc respectively, and ligated them overnight at 16°C with T4 ligase. The constructed plasmid was identified by PCR, enzyme digestion and sequencing respectively, and after confirming that the direction and sequence were correct, the extracted plasmid was linearized. The mMESSEGemMACHINE T3 in vitro transcription kit of Ambion Company was used for in vitro transcription. For details, please refer to the company's instruction manual. The resulting product wa...

Embodiment 3

[0041] Example 3 Expression of Aquaporin 2 in Anopheles sinensis at different developmental stages and in different tissues of adult mosquitoes

[0042] Collect mosquito samples from the same batch of Anopheles sinensis at different developmental stages, namely: mosquito eggs, 4th instar larvae, pupae, adults (male mosquitoes, female mosquitoes); and the collection of different tissues of Anopheles sinensis, from the same batch 50-60 female mosquitoes were taken, and the head, midgut, ovary, and fat body were separated under a dissecting microscope to extract total RNA for fluorescent quantitative PCR detection.

[0043] The collected frozen specimens were ground and homogenized on ice. Total RNA was extracted by Qiagen RNeasy kit and treated with RNase-free DNAse to remove contamination from genomic DNA. Take 2 μg of total RNA and synthesize cDNA by reverse transcription with random primers according to the instructions of the Toyobo reverse transcription kit. cDNA samples ...

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Abstract

The invention discloses an encoding gene from anopheles sinensis aquaporin 2 and an application of the anopheles sinensis aquaporin 2, belongs to the technical field of bioengineering, and provides the encoding gene of the anopheles sinensis aquaporin 2, polypeptides synthetized on the basis of the encoding gene, built recombinant protein and an application technology of the anopheles sinensis aquaporin 2 in an insect pesticide. Complementary deoxyribonucleic acid (cDNA) of the anopheles sinensis aquaporin 2 comprises 753 basic groups, has a nucleotide sequence shown in SEQ ID No.1, and is named as (i)AsAQP(/i)2; the anopheles sinensis aquaporin 2 encodes 250 amino acids, has an amino acid sequence shown in SEQ ID No.2, and has 90% of homology with the amino acid sequence (AGAP008842) corresponding to anopheles gambiae. The molecular weight of the anopheles sinensis aquaporin 2 is 26.0kDa; the isoelectric point is 6.40; the anopheles sinensis aquaporin 2 is a stable protein. The in vivo experiment proves that the anopheles sinensis aquaporin 2 has an efficient moisture transport function. 14 amino acids at N-terminal have good antigenicity, and can induce generation of immuno-antibodies. The recombinant protein has a good application prospect in the insect pesticide.

Description

technical field [0001] The present invention relates to an aquaporin and its coding gene and application, in particular to aquaporin 2 derived from Anopheles sinensis ( AsAQP 2) The coding gene and its application belong to the field of bioengineering technology. Background technique [0002] Anopheles sinensis ( Anopheles sinensis ) is an important vector of malaria and filariasis. It is not only distributed in all provinces and regions of the country except Xinjiang and Qinghai, but also widely distributed in Southeast Asia and the Western Pacific. The main malaria vector in the endemic area of ​​vivax malaria has a large population in my country. Anopheles sinensis is an important vector organism in my country, and effective control of Anopheles sinensis is of great significance to control the spread of related diseases. [0003] Insecticides are currently the main way to control Anopheles mosquitoes, but due to the wide application and dose increase of insecticides in...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C12N15/70C12N1/21
Inventor 唐建霞曹俊张超李菊林
Owner JIANGSU INST OF PARASITIC DISEASES
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