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Clonorchis sinensis GTP bindin alpha subunit, its coded nucleic acid and its use

A Clonorchis sinensis, protein-binding technology, which can be used in applications, anti-animal/human immunoglobulins, drug combinations, etc., and can solve the problems of blocked release of nerve transmitters and different

Inactive Publication Date: 2005-10-19
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Blockage of neurotransmitter release by microinjection of the GTP congener GTPγS into synaptic terminals
[0006] Rao's research shows that there are a large number of 20-30Kda GTP-binding proteins in the eye lens, and at the same time there are 33-45Kda GTP-binding proteins, but the 33-45Kda GTP-binding proteins are closely related to small GTP-binding proteins and heterologous three The aggregated GTP-binding proteins are all significantly different, and have not been found in brain\heart and other tissues. GTP-binding proteins play an important role in regulating the formation of crystallins, the growth and differentiation of lens cells, etc.

Method used

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  • Clonorchis sinensis GTP bindin alpha subunit, its coded nucleic acid and its use
  • Clonorchis sinensis GTP bindin alpha subunit, its coded nucleic acid and its use

Examples

Experimental program
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Effect test

Embodiment 1

[0098] Example 1: Cloning of Clonorchis sinensis GTP-binding protein alpha subunit

[0099] The total RNA of Clonorchis sinensis adults was extracted by one-step method of guanidine isothiocyanate / phenol / chloroform. Poly(A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (product of Qiegene). 2ug poly(A) mRNA was reverse transcribed to form cDNA. Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragment into the multiple cloning site of the pBSK(+) vector (product of Clontech Company), transform DH5α, and the bacteria formed a cDNA library. The sequences of the 5' and 3' ends of all clones were determined with Dye terminate cycle reactions sequencing kit (product of Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer). Comparing the determined cDNA sequence with the existing public DNA sequence database (Genebank), it was found that the cDNA sequence of one of the clones, 3g11, was a new DNA. The insert cDNA fragment contai...

Embodiment 2

[0100] Example 2: Homology retrieval of cDNA clones

[0101] With the sequence of the Clonorchis sinensis GTP-binding protein α subunit of the present invention and the protein sequence encoded thereof, use the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403 -10], homologous searches were performed in databases such as Genbank and Swissport. The gene with the highest homology to the polypeptide of the present invention is a known Lymnaea stagnalis GTP-binding protein α subunit, and its accession number in Genbank is p30682. The results show that the two are highly homologous, the identity is 64%; the similarity is 80%.

Embodiment 3

[0102] Example 3: Cloning of the gene encoding the Clonorchis sinensis GTP-binding protein α subunit by RT-PCR

[0103] The total RNA of Clonorchis adultes was used as template, and oligo-dT was used as primer to carry out reverse transcription reaction to synthesize cDNA. After purification with Qiagene kit, PCR amplification was carried out with the following primers:

[0104] Primer1: 5'-GGATTCCGAATTCGAGCGGTCCAT-3' (SEQ ID NO: 3)

[0105] Primer2: 5'-TTTTTCATGGTCCTAAGGGATTGATT-3' (SEQ ID NO: 4)

[0106] Primer1 is the forward sequence starting from the 1st bp at the 5' end of SEQ ID NO:1;

[0107] Primer2 is the 3' reverse sequence in SEQ ID NO:1.

[0108] The conditions of the amplification reaction: 50mmol / L KCl, 10mmol / L Tris-Cl, (pH8.5), 1.5mmol / L MgCl in a reaction volume of 50μl 2, 200 μmol / L dNTP, 10 pmol primer, 1 U of Taq DNA polymerase (product of Clontech Company). On a PE9600 DNA thermal cycler (Perkin-Elmer Company), the reaction was performed for 25 cycles...

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Abstract

The present invention discloses one new gene coding the clonorchis sinonsis GTP bindin alopha subunit, the gene coded polypeptide and the antibody of the polypeptide. The present invention also discloses clonorchis sinonsis GTP bindin alopha subunit screening inhibitor of the polypeptide and the application of the polynucleotides as primer or probe, especially the use of the polypeptide and the polynucleotides as diagnosis kit and vaccine.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the invention describes a new polypeptide-Clonorchis sinensis GTP-binding protein α subunit, and a polynucleotide sequence encoding the polypeptide. The present invention also relates to the application of this polynucleotide and polypeptide. Background technique [0002] Clonorchis sinensis is a zoonotic disease. Adults parasitize in the liver and bile ducts. Adults will destroy the biliary epithelium and submucosal blood vessels, produce and secrete excretory antigens, cause inflammation in the bile duct and around the bile duct, and lead to local dilation of the bile duct and bile duct epithelial cells. hyperplasia. Some antigen components can also enter the liver tissue through the bile duct epithelial cells, causing inflammatory response and liver function damage. Long-term severe infection can lead to fibrous tissue hyperplasia and atrophic degeneration of liver cells, and even ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K48/00A61P33/00C07K14/435C07K16/18C12N15/12C12Q1/68G01N33/569G01N33/68
Inventor 余新炳徐劲吴忠道郑南才杨光
Owner SUN YAT SEN UNIV
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