Microsome GST-1 of clonorchis sinensis, its coding acid and application

A technology of Clonorchis sinensis, GST-1, applied in the directions of application, medical preparations containing active ingredients, biochemical equipment and methods, etc.

Inactive Publication Date: 2005-09-21
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Microsomal GST-1 cDNA was isolated from rat and human liver with 83% homology, but

Method used

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  • Microsome GST-1 of clonorchis sinensis, its coding acid and application
  • Microsome GST-1 of clonorchis sinensis, its coding acid and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1: Cloning of Clonorchis sinensis microsomal GST-1

[0101] 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, c008C08, was a new DNA. The insert cDNA fragment contained in this cl...

Embodiment 2

[0102] Example 2: Homology retrieval of cDNA clones

[0103] With the sequence of the Clonorchis sinensis microsomal GST-1 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], and performed homology searches in databases such as Genbank and Swissport. The gene with the highest homology to the polypeptide of the present invention is a known mouse microsomal GST-1, and its accession number in Genbank is AAD51096.1. The results showed that the identity between the two was 37%; the similarity was 51%.

Embodiment 3

[0104] Example 3: Cloning of the gene encoding Clonorchis sinensis microsomal GST-1 by RT-PCR

[0105] 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:

[0106] Primer1: 5'-GGAAAATTCAAATTTTATTATTAG-3' (SEQ ID NO: 3)

[0107] Primer2: 5'-CATAAATCCCGCAGTATATTGAA-3' (SEQ ID NO: 4)

[0108] Primer1 is the forward sequence starting from the 1st bp at the 5' end of SEQ ID NO:1;

[0109] Primer2 is the 3' reverse sequence in SEQ ID NO:1.

[0110] 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 following conditions were followed for 25 cycles: 94°C f...

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Abstract

The invention discloses new genes for encoding clonorchis sinensis microsome GST-1, polypeptides encoded by the genes, and antibody of the polypeptides. The invention also discloses the depressor of the polypeptides for screening clonorchis sinensis microsome GST-1, the use of polynucleotides as primer or as probe, in particular the use of the polypeptide and polynucleotide as diagnosis reagent kit, vaccine and pharmaceutical composition.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the invention describes a new polypeptide-clonorchia sinensis microsome GST-1, 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 cause cancer, ...

Claims

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Application Information

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IPC IPC(8): A61K38/45A61K48/00C07K16/40C12N9/10C12N15/54C12Q1/68G01N33/53
Inventor 余新炳吴忠道徐劲陈守义吴德
Owner SUN YAT SEN UNIV
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