Application of cystatin S

A protein and capture agent technology, applied in the field of medical detection, can solve the problems of gastric cancer efficacy evaluation, metastasis and recurrence monitoring, etc., and achieve the effect of good specificity and high sensitivity

Active Publication Date: 2014-07-09
SHANGHAI LIANGRUN BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there have been no reports that CST4 can be used for the evaluation of the efficacy of gastric cancer and the monitoring of metastasis and recurrence, and there are no reports about CST4 and other diseases such as pancreatic cancer, esophageal cancer, liver cancer, and gastrointestinal stromal tumors.

Method used

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  • Application of cystatin S
  • Application of cystatin S
  • Application of cystatin S

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation of recombinant plasmid standard

[0035] Recombinant plasmid standard preparations were prepared according to the steps and conditions in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al., translated by Huang Peitang et al., Science Press, 2002):

[0036] Primers for amplifying the truncated fragment of the CST4 gene were designed according to the reported CST4 gene sequence (the nucleotide sequence of the CST4 gene is shown in SEQ ID NO.1), as follows:

[0037] Upstream primer: 5'-ctgcctctgaggagaccaccatggc-3' (SEQ ID NO.2);

[0038] Downstream primer: 5'-ggcgatgctactgtttaattgcag-3' (SEQ ID NO.3).

[0039]Use the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3 as primers, and human cDNA as a template for PCR amplification. PCR reaction system: the final concentration of upstream and downstream primers is 250nM, and the final concentration of Taq DNA polymerase 1U / reaction; the final concentration of dNTPs was 250...

Embodiment 2

[0040] Example 2 Construction of CST4 fluorescent quantitative PCR detection kit

[0041] Design primers and probes for quantitative detection of CST4 gene, CST4 gene cDNA, CST4 gene mRNA or CST4 gene truncated fragments according to the CST4 gene sequence, as follows:

[0042] The upstream primer is: 5'-aacaaggccaccgaagatga-3' (SEQ ID NO.5); the downstream primer is: 5'-ccacctctacgtcgaagaagta-3' (SEQ ID NO.6); the probe is: FAM-cccaaaggtctgctccctggctcgca-TAMRA (SEQ ID NO.7). At the same time, the human B2M gene is used as the internal control, and the detection primers and probes of the internal control gene are designed: the upstream primer is: 5'-actgaattcacccccactga-3' (SEQ ID NO.8); the downstream primer is: 5'-cctccatgatgctgcttaca-3' ( SEQ ID NO.9); the probe is: FAM-tatgcctgccgtgtgaaccatgtgac-TAMRA (SEQ ID NO.10). Then, the designed primers and probes were used together with other conventional reagents to form a CST4 fluorescence quantitative detection kit. The compon...

Embodiment 3

[0062] Example 3 Establishment of Cystatin S ELISA reaction system and its optimization

[0063] Coat the ELISA plate with a mouse anti-human Cystatin S monoclonal antibody at a concentration of 5 μg / mL, coat it overnight at 4°C, and wash the plate; then block it with 2% BSA at room temperature for 2 hours, and wash the plate; Respectively 0pg / mL, 50pg / mL, 100pg / mL, 200pg / mL, 400 pg / mL, 800 pg / mL, 1600 pg / mL Cystatin S protein standard (the amino acid encoding Cystatin S protein is shown in SEQ ID NO. 11) and samples were added to the closed plate, reacted at 37°C for 1 hour, and washed the plate; then hybridized with rabbit anti-human Cystatin S polyclonal antibody labeled with HRP at a concentration of 0.5 μg / mL, and reacted at 37°C for 1 hour , after washing the plate, react with tetramethylbenzidine (TMB) for 2-3 minutes, and finally stop the reaction with 2M sulfuric acid, and detect the OD value under the condition of 450nm, and then take the Cystatin S protein standar...

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Abstract

The invention discloses new application of cystatin S, and specifically discloses application of cystatin S to prepare a marker for diagnosing and indicating liver cancer, gastrointestinal stromal tumor, pancreas cancer, esophagus cancer or stomach cancer. The invention also discloses a trapping agent of the marker for diagnosing and indicating liver cancer, gastrointestinal stromal tumor, pancreas cancer, esophagus cancer or stomach cancer, and the trapping agent is applied to prepare a kit for diagnosing and indicating liver cancer, gastrointestinal stromal tumor, pancreas cancer, esophagus cancer or stomach cancer. The prepared kit has the advantages of being good in specificity, high in sensitivity and the like, is applicable to early diagnosis on liver cancer, gastrointestinal stromal tumor, pancreas cancer, esophagus cancer or stomach cancer, assessment on treatment effect during treatment and monitoring on metastasis and recurrence after treatment is finished.

Description

technical field [0001] The invention belongs to the field of medical detection and relates to the application of cysteine ​​protease inhibitor S. Background technique [0002] With the deepening of the research on the pathogenesis of tumors, it is found that the Cystatin family, as cathepsins, are endogenous inhibitors of cysteine ​​proteases, and play a very important role in the occurrence, development, invasion and metastasis of tumors. The results of the study showed that the expression levels of Cystatin family members increased in different tumors. For example, the expression levels of Cystatin C increased in different levels in ovarian cancer and head and neck cancer, and the expression levels of stefin A in non-small cell lung cancer increased. The expression level of Cystatin F in a variety of tumors has increased significantly, which is probably due to the need for the participation of cathepsin in the process of tumorigenesis and development, which first induces a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C07K16/38G01N33/68
CPCC12Q1/6886C12Q2600/106C12Q2600/118G01N33/57438G01N33/57446G01N33/57484G01N2333/8139
Inventor 王弢秦勇渠香云
Owner SHANGHAI LIANGRUN BIOMEDICINE TECH CO LTD
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