Application of cystatin (SN)

A protein and capture agent technology, which is applied in the field of medical detection, can solve the problems that have not been reported in the literature, and achieve the effect of high sensitivity and good specificity

Active Publication Date: 2014-07-02
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, the relationship between CST1 and other cancers, such as pancreatic cancer, esophageal

Method used

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  • Application of cystatin (SN)
  • Application of cystatin (SN)
  • Application of cystatin (SN)

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0032] Example 1 Preparation of recombinant plasmid standard

[0033] Recombinant plasmid standard preparations were prepared according to the steps and conditions described in the Molecular Cloning Experiment Guide (third edition, J. Sambrook et al., translated by Huang Peitang et al., Science Press, 2002):

[0034] According to the sequence of CST1 gene (the nucleotide sequence of CST1 gene is shown in SEQ ID NO.1), the truncated fragment of CST1 gene was designed and amplified, as follows:

[0035] Upstream primer: 5'-tcgggctctcaccctcctct-3' (SEQ ID NO. 2);

[0036] Downstream primer: 5'-agggaggcgatgctactgttta-3' (SEQ ID NO. 3).

[0037] Use the nucleotide sequences shown in SEQ ID NO. 2 and SEQ ID NO. 3 as primers and human cDNA as template for PCR amplification. PCR reaction system: the final concentration of the upstream and downstream primers is 250 nM, and the final concentration of Taq DNA polymerase It is 1U / reaction; the final concentration of dNTPs is 250 nM. Refer to the k...

Example Embodiment

[0038] Example 2 Construction of CST1 fluorescent quantitative PCR detection kit

[0039] According to the nucleotide sequence of CST1 gene, design the absolute quantitative PCR primers and probes for detecting CST1 gene, CST1 gene cDNA, CST1 gene mRNA or CST1 gene truncated fragment, as follows:

[0040] The upstream primer is: 5'-ggtactaagagccaggcaacag-3' (SEQ ID NO. 5); the downstream primer is: 5'-agttgggctgggacttggta-3' (SEQ ID NO. 6); the probe is: FAM-cggcccacctctacgtcgaagaagtaattca-TAMRA (SEQ ID NO.7). At the same time, human B2M gene is used as the internal control, and the detection primers and probes of the internal control gene are designed: upstream primer: 5'-actgaattcacccccactga-3' (SEQ ID NO. 8); downstream primer: 5'-cctccatgatgctgcttaca-3' (SEQ ID NO .9); Probe: FAM -tatgcctgccgtgtgaaccatgtgac- TAMRA (SEQ ID NO.10). Then, the designed primers and probes and other conventional reagents are used to form a CST1 mRNA absolute quantitative detection kit. The componen...

Example Embodiment

[0057] Example 3 Establishment and optimization of Cystatin SN serum detection reaction system

[0058] Coat the ELISA plate with the mouse anti-human Cystatin SN monoclonal antibody at a concentration of 5μg / mL, coat it overnight at 4°C, and wash the plate; then block in 2% BSA at room temperature for 2 hours and wash the plate; Cystatin SN protein standard products with concentrations of 0 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 400 pg / mL, 800 pg / mL, 1600 pg / mL (the amino acid sequence encoding the Cystatin SN protein is shown in SEQ ID No. 11) and the sample were added to the closed plate, reacted at 37°C for 1 hour, and the plate was washed; then, the rabbit anti-human Cystatin SN polyclonal antibody labeled with HRP at a concentration of 0.5μg / mL was used to react at 37°C 1 After hours, wash the plate; then react with tetramethylbenzidine (TMB) for 2-3 minutes, finally stop the reaction with 2M sulfuric acid, and detect the OD value at 450nm ( Image 6 ). by Image 6 It can...

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Abstract

The invention discloses novel application of cystatin (SN), and particularly relates to application of cystatin SN to preparation of markers for diagnosing and indicating liver cancer, gastrointestinal stromal tumor, pancreas cancer or esophagus cancer. The invention further discloses a trapping agent of the markers for diagnosing and indicating liver cancer, gastrointestinal stromal tumor, pancreas cancer or esophagus cancer, and application of the trapping agent to preparation of a kit for diagnosing and indicating liver cancer, gastrointestinal stromal tumor, pancreas cancer or esophagus cancer. The prepared kit has the advantages of good specificity, high sensitivity and the like, and can be used for early diagnosing liver cancer, gastrointestinal stromal tumor, pancreas cancer or esophagus cancer, evaluating the treatment effect in the treatment process, and monitoring the transfer relapse after treatment.

Description

technical field [0001] The invention belongs to the field of medical detection and relates to the application of cysteine ​​protease inhibitor SN. 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 ...

Claims

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Application Information

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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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