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Kit for early detection of liver cancer and preparation method thereof

A technology for early detection and kits, applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of complex preparatory work for chemical cross-linking, reduced enzyme activity, and many cross-linked by-products, etc., to achieve good targeting High sensitivity, less by-products, and high sensitivity

Inactive Publication Date: 2018-12-25
CHINA UNIV OF PETROLEUM (EAST CHINA) +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using chemical cross-linking technology to label phycobiliproteins and enzymes, the activity of enzymes may be reduced after the reaction, and the preparatory work for chemical cross-linking is complicated, requiring separate purification of various cross-linking raw materials, and there are many cross-linking by-products

Method used

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  • Kit for early detection of liver cancer and preparation method thereof
  • Kit for early detection of liver cancer and preparation method thereof
  • Kit for early detection of liver cancer and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 expresses the construction of recombinant protein PCBS or PEBS genetic engineering strain

[0050] According to the gene sequence related to the phycobiliprotein biosynthesis pathway in the Spirulina genome published in the international gene database, relevant primers were designed to clone the key enzyme genes cpcA, cpcE / F, hox1, pcyA, pebS, etc. for phycobiliprotein synthesis. Streptavidin gene sa deduced its nucleotide sequence according to the amino acid sequence published in NCBI, and obtained it by chemical synthesis after codon optimization. The streptavidin gene sa is shown in SEQ ID NO: 1, and the codon-optimized streptavidin gene sa is shown in SEQ ID NO: 2. The optimized sa sequence can improve the soluble expression of SA in Escherichia coli and increase its expression level.

[0051] The present invention uses bioengineering strains to stably produce the method of phycobiliprotein α subunit fused with streptavidin with biotin binding ability ...

Embodiment 2

[0066] Embodiment 2 Expression purification and characterization of recombinant expression plasmid PCBS or PEBS

[0067] (1) Recombinant expression plasmid PCBS or PEBS shake flask culture

[0068] The recombinant expression plasmid PCBS or PEBS is transformed into Escherichia coli BL21(DE3) to obtain engineering strain P1 or P2. The conventional fermentation engineering method is used for cultivation, the optimized induction conditions are used to induce the expression of the recombinant protein, and the optimized preparation method is used for the purification and preparation of the fusion protein. In the induction conditions described above, the overnight cultured seed solution was added to 1L of TB medium (containing TB medium 900mL, TB salt 100mL) at a volume ratio of 1%, and the bacteria were cultured on a constant temperature shaker at 37°C at 170rpm to OD 600 When it reaches 1.3, add one or both of the inducer IPTG and lactose, the final concentration of the inducer i...

Embodiment 3

[0075] Example 3 Preparation of a kit for early detection of liver cancer

[0076] A preparation method of a kit for early detection of liver cancer, the specific steps are as follows:

[0077] (1) Add 100 μL of 5 μg / mL monoclonal antibody against liver cancer markers to each well of the fluorescent microtiter plate, and absorb overnight at 4°C;

[0078] (2) Shake and wash with washing liquid at 170r / min for 3min, repeat 3 times;

[0079] (3) Add 200 μL of blocking solution to each well, and incubate for 120 min in a 37°C constant temperature incubator;

[0080] (4) Shake and wash with washing liquid at 170r / min for 3min, repeat 3 times;

[0081] (5) Add 100 μL each of the sample to be tested, the negative control PBST solution, and the positive control liver cancer marker AFP into the reaction well, repeat 3 times for each well, and cover the fluorescent microplate cover;

[0082] (6) Shake and wash with washing liquid at 170r / min for 3 minutes, shake off the liquid in the...

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Abstract

The invention discloses a kit for early detection of liver cancer and a preparation method thereof. The kit comprises a fluorescent enzyme labeling board, sample diluent, washing liquid, a negative control, a positive control, a monoclonal antibody against the liver cancer markers, a biotinylated monoclonal antibody against the liver cancers marker and a streptavidin-phycobiliprotein fluorescent probe. The kit for early detection of liver cancer reduces the step of adding chemical substrate for color development, the operation steps are simple, the sensitivity is high, and the by-products aresmall. The kit for early detection of liver cancers and preparation method thereof realizes the synthesis of streptavidin / phycobiliprotein fusion fluorescent protein by "in vivo one-step" in Escherichia coli by multi-gene combined expression. When the phycobiliprotein labeling is completed, the fusion molecule is guaranteed to have stable fluorescence properties. At the same time, the fusion protein also has good targeting to biotinylated substances, thereby enhancing the application ability of phycobiliprotein in immunoassay.

Description

technical field [0001] The invention relates to a kit for early detection of liver cancer and a preparation method thereof, belonging to the technical field of liver cancer detection. Background technique [0002] Liver cancer, especially primary liver cancer, is one of the most common malignant tumors in my country and ranks second in cancer deaths. About 130,000 people die of liver cancer in my country every year, accounting for more than half of the new cases of liver cancer in the world. Most liver cancer patients are in the middle or late stage when they see a doctor, and miss the best time for treatment. Early diagnosis and early treatment are the most effective ways to prevent and treat liver cancer and reduce mortality. At present, the liver cancer detection kits used in the market mostly use chemiluminescence method for detection. This method has the disadvantages of high equipment requirements, complicated operation steps, easy interference of experimental results ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/533
CPCG01N33/533G01N33/68
Inventor 葛保胜王祥法黄方迟海霞
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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