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Japanese blood fluke cell periodic static agent, its coded nucleic acid and its use

A cell cycle, schistosomiasis technology, applied in applications, medical preparations containing active ingredients, animal/human peptides, etc., can solve problems such as non-recognition

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

This protein has only 30% homology with human stathmin and cannot be recognized by anti-human stathmin antibodies

Method used

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  • Japanese blood fluke cell periodic static agent, its coded nucleic acid and its use
  • Japanese blood fluke cell periodic static agent, its coded nucleic acid and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1: Cloning of Schistosoma japonicum cell cyclin

[0095] The full-length library was constructed using the "SMART" cDNA library construction kit.

[0096] Take the library solution and dilute it to 1:100, pre-adsorb with the phage host bacteria XL1-Blue for 15 minutes, add it to the top agar, and spread it on a plate containing LB and 10mM MgSO4, after 6-18 hours (see the phage The spots are fused with each other), add SM solution to elute overnight, absorb the eluent and centrifuge at 4000g for 5 minutes, absorb the supernatant and add 20% PEG8000, after half an hour to precipitate DNA, centrifuge at 12000g for 15 minutes, boil and dissolve with Mini Q water, and use universal primers Perform PCR amplification. PCR reaction system (25 μl total reaction): 2.5 μl of 10×buffer (containing Mg2+), 0.5 μl of 10 mM dNTP, 0.5 μl of each primer, 10.0 μl of library DNA template, 0.25 μl of Taq enzyme, and water up to 25.0 μl. The reaction parameters are: pre-denaturati...

Embodiment 2

[0097] Example 2: Homology retrieval of cDNA clones

[0098] With the sequence of Schistosoma japonicum cell cyclin of the present invention and its encoded protein sequence, use the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10], Homology 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 Schistosoma mannensis tathmin-like Protein, and its accession number in Genbank is AF091509. Blastx results showed that the identity of the two was 100%.

Embodiment 3

[0099] Example 3: Cloning of the gene encoding Schistosoma japonicum cell cyclin by RT-PCR

[0100] According to the instructions of Trizol Reagent, the total RNA of Schistosoma japonicum adults was extracted, and the following primers were used to carry out RT-PCR (one-step method) according to the instructions of Promega AccessRT-PCR:

[0101] Primer1: 5'-ggcttccagtaaaatgaaagt-3' (SEQ ID NO: 3)

[0102] Primer2: 5'-gagttttcatcaatctttatt-3' (SEQ ID NO: 4)

[0103] Primer1 is the forward sequence starting from the 1st bp at the 5' end of SEQ ID NO:1;

[0104] Primer2 is the 3' reverse sequence in SEQ ID NO:1.

[0105] 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 PE9600 type DNA thermal cycler (Perkin-Elmer Company), the reaction was carried out for 25 cycles according to the following conditio...

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Abstract

The present invention discloses one new gene coding Japanese blood fluke cell periodic arrest prime, the gene coded polypeptide and the antibody of the polypeptide. The present invention also discloses Japanese blood fluke cell periodic arrest prime 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-Schistosoma japonicum cell cycle quiescent factor, and a polynucleotide sequence encoding the polypeptide. The present invention also relates to the application of this polynucleotide and polypeptide. Background technique [0002] Schistosomiasis is still one of the serious public health problems today. Vaccines can be used to prevent and treat schistosomiasis. Research and finding a suitable anti-schistosomiasis vaccine has become a research hotspot. In China, there are six candidate vaccine molecules for schistosomiasis mansoni recommended by WHO-Sj 28 GST, paramyosin, triose phosphate isomerase, integral membrane protein, irradiation attenuated antigen-5 and fatty acid binding protein, and Sj32 , cathepsin B, tropomyosin, vitelloferritin, LDH, Sj16, and CyPA, etc., observed that their protective effects were unsatisfactory. Therefore, the s...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61K48/00A61P33/12C07K14/435C07K16/18C12N15/12C12Q1/68G01N33/569G01N33/68
CPCY02A50/30
Inventor 吴忠道余新炳胡旭初阮志燕胡少敏孟玮
Owner SUN YAT SEN UNIV
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