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Pepsinogen II recombinant protein and its monoclonal antibody, preparation method and application thereof

A pepsinogen and monoclonal antibody technology, applied in the biological field, can solve the problems of high cost, labor and labor, etc., and achieve the effects of sufficient supply, cost reduction, and simple quantification

Inactive Publication Date: 2021-06-29
黎榕萍
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the first method is laborious and laborious, the cost is relatively high, and the second method also needs to consider a suitable expression system so that the expressed protein is closer to the PG II protein in the human body

Method used

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  • Pepsinogen II recombinant protein and its monoclonal antibody, preparation method and application thereof
  • Pepsinogen II recombinant protein and its monoclonal antibody, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The preparation of embodiment 1 pepsinogen II

[0020] The codon-optimized nucleotide sequence of pepsinogen II (the specific sequence is shown in SEQ ID NO.1) is cloned into a eukaryotic expression vector (such as ). Using the codon-optimized nucleotide sequence of pepsinogen II (the specific sequence is shown in SEQID NO.1) as a template, EcoRI and Hind III double restriction sites were used to connect to construct pEE12.4-PGII-6His expression plasmid. The recombinant plasmids identified as positive were sent to Huada Biological Company for sequence determination, and software was used to analyze and compare the determined nucleotide sequence and encoded amino acid sequence to check the correctness of the reading frame.

[0021] The correctly identified pEE12.4-PGⅡ-6His expression plasmid was transfected into CHO-K1 cells. Pressurized screening started 24 hours after transfection: Take out the six-well plate cells from the 37°C incubator, discard the supernatant med...

Embodiment 2

[0024] Example 2 Preparation of anti-pepsinogen II monoclonal antibody

[0025] The pepsinogen II recombinant protein prepared in Example 1 was used to immunize 8-week-old BALB / c mice. For the first immunization, the pepsinogen II recombinant protein was emulsified with an equal volume of Freund's complete adjuvant, and the mice were inoculated intraperitoneally, with 100 μg of protein 7 days later, pepsinogen II recombinant protein was emulsified with equal volume of Freund's incomplete adjuvant, and the mice were immunized by intraperitoneal inoculation for the second time, 100 μg protein / mouse; 7 days later, the mice were directly immunized with pepsin for the third time by intraperitoneal route Original Ⅱ recombinant protein, 100 μg / mouse; on the 3rd day after immunization, take mouse splenocytes to fuse with mouse myeloma cell SP2 / 0, and culture in HAT selective medium; 10 days later, use pepsinogen Ⅱ recombinant protein as a package The cell supernatant was detected by a...

Embodiment 3

[0028] Example 3 Application of pepsinogen Ⅱ and anti-pepsinogen Ⅱ monoclonal antibody

[0029] Using the PGII protein prepared by the invention as a calibrator and two monoclonal antibodies prepared by the invention as detection antibodies, a set of diagnostic reagents for the PGII protein is established. For the specific reagent preparation method, see CN 109307765 A. Use reagent of the present invention and Abbott's test kit to carry out performance comparison, the result is as shown in the table below: the diagnostic reagent prepared with PGⅡ protein of the present invention and monoclonal antibody can be used in the detection of PGⅡ protein, and detection result is very good (specificity good, high sensitivity), and the coincidence rate with imported reagents is as high as 96.8%.

[0030]

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Abstract

The invention discloses a CHO-K1 cell strain, which contains a gene capable of efficiently secreting and expressing PG II recombinant protein. The invention also discloses a method for preparing the CHO-K1 cell strain, and the method comprises the following steps: 1) cloning a gene sequence as shown in SEQ ID NO.1 into an eukaryotic expression vector to obtain a recombinant plasmid containing the gene encoding the PG II recombinant protein; (2) transfecting the recombinant plasmid into a CHO-K1 cell, so as to obtain a CHO-K1 cell strain; and 3) culturing, screening and domesticating the CHO-K1 cell strain in the step 2) to obtain the cell strain capable of efficiently secreting and expressing the PG II recombinant protein. The invention also discloses an anti-PG II monoclonal antibody, wherein the antigen epitope combined with a monoclonal antibody 1 is located at aa78-aa90 of the pepsinogen II; and the antigen epitope combined with a monoclonal antibody 2 is located at aa280-aa291 of the pepsinogen II. The protein provided by the invention is high in expression quantity, good in quality and low in cost; the monoclonal antibody can be paired and used for detecting the PG II protein, and is good in specificity and high in sensitivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a pepsinogen II recombinant protein, a monoclonal antibody thereof and a preparation method thereof. Background technique [0002] Pepsinogen (PG) is an endoprotease with digestive function. It is a single-chain polypeptide composed of 375 amino acids and belongs to the aspartic acid protein family. Human pepsinogen can be divided into pgl-pg7 seven isoenzymes according to the mobility under agar gel electrophoresis from fast to slow, among which pgl-pg5 has a common immunogenicity, also called PG I; pg6 to pg7 Slow mobility, also called PG II. In vivo, PG I and PG II differ in their cellular origin and tissue distribution. Studies have shown that the decrease of PG I level is a reliable sign of the occurrence and development of gastric disease. Some people in foreign countries regard low levels of serum PG as a sign of the occurrence and development of gastric disease...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N9/64C12N15/85C07K16/40G01N33/573
CPCC07K16/40C12N9/6481C12N15/85C12Y304/23002G01N33/573
Inventor 黎榕萍
Owner 黎榕萍
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