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Preparation method of novel anti-human papilloma viral protein

An anti-human papilloma and virus technology, applied in the direction of antiviral agents, biochemical equipment and methods, viruses, etc., can solve the problems of not meeting the market demand, low expression of natural PAP protein, and lack, etc., to achieve large-scale application Prospect, simple preparation method, effect of reducing operation steps

Pending Publication Date: 2019-03-26
江苏正大天创生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main technical problems in the prior art are as follows: the expression of natural PAP protein is very small, which cannot meet the market demand
There is a lack of a method that can produce a large amount of PAP protein in vitro, and the PAP protein that needs to be produced has the same biological activity as the natural PAP protein

Method used

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  • Preparation method of novel anti-human papilloma viral protein
  • Preparation method of novel anti-human papilloma viral protein
  • Preparation method of novel anti-human papilloma viral protein

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0025] The preparation method of novel anti-human papillomavirus protein of the present invention comprises the following steps:

[0026] 1. According to the sequence of the PAP gene published on Genbank, the coding region of the PAP gene is 942bp in length, encoding 313 amino acids, with a signal peptide consisting of 22 amino acids at the N-terminus and a stable region consisting of 29 amino acids at the C-terminus . The amplification of the gene is used for the construction of the later eukaryotic expression vector, so after removing the 22 amino acids encoded by the 66 bases at the N-terminus, the total length is 876bp. The N-terminal mutated PAP gene sequence was synthesized, primers were designed with Prime 5.0 software, and the N-terminal mutated PAP gene was used as a template for PCR amplification. After the PCR product was recovered, it was connected to the secreted expression vector pPICk9 vector to obtain the recombinant plasmid pPICk9-PAP, and The Pichia pastoris...

experiment example 2

[0032] Experimental Example 2 Determination of the concentration of recombinant protein prepared in Example 1

[0033] BCA Micropore Quantitative Detection Method:

[0034] (1) Dilute the BSA standard: Dilute the BSA standard with the diluent consistent with the protein sample to be tested as shown in the table below.

[0035]

[0036] (2) Configure BCA working fluid:

[0037] ① Calculate the total amount of BCA working fluid:

[0038] Total amount of BCA working solution = (number of BSA standard samples + number of unknown samples) × number of multiple wells × volume of BCA working solution for each sample

[0039] The microwell detection method generally uses 3 duplicate wells, and the volume of BCA working solution for each sample is 200 μL

[0040] ②According to the calculated total amount of BCA working solution required, prepare BCA working solution with BCA-A and BCA-B according to the volume ratio of 50:1, and mix well.

[0041] (3) Quantitative detection:

[...

experiment example 3

[0048] Experimental Example 3 Western blot analysis of natural PAP protein and recombinant PAP protein

[0049] The natural and the recombinant PAP protein prepared in Example 1 were subjected to SDS-PAGE electrophoresis. After the electrophoresis, the gel was placed on the transfer buffer for rinsing, and the protein was transferred from the gel to the nitrocellulose membrane (NC); Stain the NC membrane with red, and then use PBST to wash off the color; transfer the membrane to the blocking solution to block; place the NC membrane in the previous step in the primary antibody diluted with PBST and incubate at 37°C for 1 hour, and then wash with PBST three times; Put the NC membrane from the previous step into the secondary antibody diluted with PBST, incubate at room temperature for 1 h, and then wash three times with PBST; under dark conditions, place the NC membrane in alkaline phosphatase containing NBT and BCIP for color development, and then Rinse with water to terminate ...

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Abstract

The invention provides a preparation method of a novel anti-human papilloma viral protein. The preparation method comprises the following steps: 1) finding a known sequence of an N-end-mutant PAP genefrom each biological database, and replacing a low-frequency used codon of Pichia pastoris in the sequence of the PAP gene with a high-frequency used codon of Pichia pastoris; artificially synthesizing a gene sequence according to the codon-modified gene sequence, wherein the N-end-mutant PAP gene is a PAP gene of which 22 amino acid sequences at the N end are removed, and the gene sequence is shown as SEQ ID NO.1; 2) connecting the artificially synthesized gene sequence to a secretory expression vector pPICk9; and 3) electrically transferring a recombinant expression vector pPICk9-PAP into Pichia pastoris, and inducing the expression of a recombinant PAP protein by using methanol. The method provided by the invention can be used for mass production of the recombinant PAP protein in vitroby utilizing the high-density fermentation of Pichia pastoris so as to be beneficial to the large-scale production of the PAP protein.

Description

technical field [0001] The invention relates to an anti-human papillomavirus protein in the technical field of medicine, in particular to a preparation method of a novel anti-human papillomavirus protein. Background technique [0002] Human papillomavirus (human papilloma virus, HPV) is a minimal DNA (deoxyribonucleic acid) virus. HPV is spherical, with a diameter of about 45-55nm (nanometer), and is epitheliophilic. HPV is a general term for a group of viruses. So far, more than 70 subtypes of HPV have been identified, and more than 100 HPV subtypes have been reported. Different subtypes can cause different diseases. According to different types of HPV and the risk of tumor occurrence, HPV can be divided into low-risk type and high-risk type. There are 14 high-risk HPV subtypes that cause cervical cancer and its precancerous lesions, namely: HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68. Among them, the two strains of H...

Claims

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Application Information

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IPC IPC(8): C07K14/025C12N15/81A61K38/16A61P31/20
CPCA61P31/20C12N15/815C07K14/005A61K38/00C12N2710/20022
Inventor 吴凯李思慧魏赵延林伟强曹丹枫朱庆平徐飞
Owner 江苏正大天创生物工程有限公司
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