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Thermostable type cytoplasm malic dehydrogenase of clonorchis sinensis, its coding nucleic acid and application

A technology of malate dehydrogenase and Clonorchis sinensis, applied in the application field of polynucleotides and polypeptides, can solve the problems of lack of catalytic and structural regulation mechanisms

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

However, cMDH lacks catalytic and structural regulatory mechanisms, and only exhibits a large number of substrate inhibition effects (Trejo F, et al. Gene172 (1996) 303-308)

Method used

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  • Thermostable type cytoplasm malic dehydrogenase of clonorchis sinensis, its coding nucleic acid and application
  • Thermostable type cytoplasm malic dehydrogenase of clonorchis sinensis, its coding nucleic acid and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1: Cloning of heat-stable cytoplasmic malate dehydrogenase from Clonorchis sinensis

[0100] 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, 2H05, was a new DNA. The insert cDNA...

Embodiment 2

[0101] Example 2: Homology retrieval of cDNA clones

[0102] With the sequence of Clonorchis sinensis thermostable cytoplasmic malate dehydrogenase of the present invention and the protein sequence encoded thereof, use Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990 ;215:403-10], homologous search in Genbank, Swissport and other databases. The gene with the highest homology to the polypeptide of the present invention is a known human malate dehydrogenase, whose accession number in Genbank is AAH01484.1, and the identity between the two is 62%; the similarity is 80%.

Embodiment 3

[0103] Example 3: Cloning of the gene encoding Clonorchis sinensis heat-stable cytoplasmic malate dehydrogenase by RT-PCR

[0104] 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:

[0105] Primer1: 5'-GGGGACACACGAGGTCAGTCGAGC-3' (SEQ ID NO: 3)

[0106] Primer2: 5'-TGCTAATAAAAGGCATCATATTATAA-3' (SEQ ID NO: 4)

[0107] Primer1 is the forward sequence starting from the 1st bp at the 5' end of SEQ ID NO:1;

[0108] Primer2 is the 3' reverse sequence in SEQ ID NO:1.

[0109] 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), the reaction was performed for 25 cy...

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Abstract

The invention discloses new genes for encoding clonorchis sinensis thermostable type cytoplasm malate dehydrogenase, polypeptides encoded by the genes, and antibody of the polypeptides. The invention also discloses the depressor of the polypeptides for screening the clonorchis senensis thermostable type cytoplasm malate dehydrogennase, the use of polynucleotides as primer or as probe, in particular the use of the polypeptide and polynucleotide as diagnosis reagent kit, and vaccine.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the invention describes a new polypeptide-clonorchia sinensis heat-stable cytoplasmic malate dehydrogenase, 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 c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/44A61K48/00C07K16/40C12N9/04C12N15/53C12Q1/68G01N33/53
Inventor 余新炳徐劲吴忠道郑南才杨光
Owner SUN YAT SEN UNIV
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