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Preparation method and application of nasopharyngeal carcinoma-relevant predisposing gene YH1 protein

A related protein, nasopharyngeal cancer technology, applied in microorganism-based methods, biochemical equipment and methods, peptide/protein components, etc., can solve problems such as no systematic report on bactericidal effect, no systematic research on the relationship between YH1 proteins, etc.

Inactive Publication Date: 2012-07-04
广东医学院司法鉴定中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is no systematic study on the relationship between YH1 protein and exogenous pathogenic microorganisms
Since the protein is secreted outside the cell, its binding to exogenous microorganisms or its bactericidal effect has not been systematically reported

Method used

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  • Preparation method and application of nasopharyngeal carcinoma-relevant predisposing gene YH1 protein
  • Preparation method and application of nasopharyngeal carcinoma-relevant predisposing gene YH1 protein
  • Preparation method and application of nasopharyngeal carcinoma-relevant predisposing gene YH1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 : target gene YH1 Acquisition, identification of positive clones and construction of expression vectors

[0037] The sequence of the YH1 protein is shown below, where the 1-19 amino acids in bold italics are the signal peptide, and according to our experience in expression and purification, the signal peptide was removed when constructing the expression vector. Total RNA was extracted from nasopharyngeal tissue of patients with chronic nasopharyngitis, cDNA was reverse-transcribed with random primers, and cDNA with BglII / Xho1 double restriction sites was used YH1 PCR amplification with specific primers YH1 Gene( figure 1 ), the primer sequence is as follows (italics are restriction sites, bolded are protected bases): NPCRP-BGL2-F: 5’- CCG AGATCT ATGCAGTTTGGAGGCCTGCCCGTG-3' (SEQ ID NO.1); NPCRP-XHO-R: 5'- CCG CTCGAG TTAGACCTTGATGACAAACTGTAG-3' (SEQ ID NO. 2). The recovered product was digested with BglII / Xho1. At the same time, the pGEX...

Embodiment 2

[0043] Example 2 : Construction of expression engineered bacteria and induced expression

[0044] Extract the correct plasmid for sequencing and dissolve it in an appropriate amount of TE solution for later use. The plasmid was transformed into BL21(DE3) expression strain. Specific method: Take out the E.coli BL21 ( DE3 ) competent cells frozen at -80°C, place them on ice immediately, and transform after thawing; take 1ul of the plasmid and add it to 100uL of competent cells, mix gently, and place in the Incubate on ice for 30 min, heat shock at 42°C for 90 seconds, immediately place on ice for 3 min; add 500ul LB liquid anti-antibiotic medium preheated at 37°C, shake at 37°C, 200 rpm for 50min; take 100-200μl of bacterial solution and spread on On the LB plate containing Amp (50 μg / ml), put it into a 37°C incubator and cultivate overnight.

[0045] Take a single colony and inoculate it in 2ml LB liquid medium (containing 50 μg / ml Amp), shake and culture at 200r / min at ...

Embodiment 3

[0047] Embodiment 3: the purification of fusion protein GST-YH1

[0048] The culture supernatant was collected by centrifugation at 5000g at 4°C, then resuspended in sonication buffer (PBS, 1%Triton X-100, 1mM EDTA, pH7.4), and the lysate was sonicated 200 times at a power of 400W on ice ( Sonicate for 3 seconds at intervals of 5 seconds), then centrifuge at 15,000g for 30 minutes and take the supernatant.

[0049]Affinity chromatography: Glutathione FF chromatography column was equilibrated with buffer (PBS, 1%Triton X-100, 1mM EDTA, pH7.4) for 10 column volumes, and then the bacterial supernatant was loaded onto the chromatography column, and then buffer ( PBS, 1mM EDTA, pH7.4) was washed for 20 column volumes, and then GST-YH1 was eluted with buffer (PBS, 10mM GSH, 1mM EDTA, pH8.0). The induced supernatant, precipitate and eluate were taken respectively for SDS-PAGE electrophoresis analysis ( Figure 6 ). The purified GST-YH1 fusion protein was stored at -80 ℃.

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Abstract

The invention provides an application of a YH1 protein to combination and agglutination of pathogenic microorganisms, provides an application of the YH1 protein to preparation of a medicament for treating inflammation-relevant diseases caused by bacterial infection, and discloses a preparation method of the YH1 protein. The method comprises the following steps of: constructing an expression vector; constructing expression engineering bacteria; and performing induced expression, purification and enzyme digestion. A recombinant YH1 protein obtained with the method has bioactivity. According to the recombinant YH1 protein, pathogenic microorganisms can be combined and agglutinated, so that the YH1 protein plays a role in killing gram negative bacteria, and can be used for preventing and treating inflammation caused by bacterial infection.

Description

technical field [0001] The invention relates to a protein preparation method of nasopharyngeal carcinoma-related susceptibility gene YH1 and the application of the protein in microorganism identification and antibacterial aspects, which can be used for inflammation-related diseases caused by bacteria. Background technique [0002] Nasopharyngeal carcinoma (NPC) is a polygenic hereditary disease involving multiple factors (Epstein-Barr virus infection, environmental factors and genetic susceptibility, etc.). Among the known oncogenes, ras , c-myc and bcl-2 Molecules such as NPC have increased expression levels in most nasopharyngeal carcinomas, but no gene structure rearrangement, mutation or gene amplification has been found, which indicates that their protein products may be involved in a certain stage of the development of nasopharyngeal carcinoma. may not play a major role in its development. Among known tumor suppressor genes, p53 The role in nasopharyngeal carcino...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61P31/04C12N15/70C12R1/19
Inventor 何志巍刘辉
Owner 广东医学院司法鉴定中心
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