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Recombinant plasmid expressing soluble human papillomavirus 16 subtype l1 protein and its expression method

A human papilloma virus and recombinant plasmid technology, applied in the field of genetic engineering, can solve the problem of unutilized cervical cancer vaccine, etc., and achieve the effects of increasing blood coagulation value, promoting correct folding, and improving efficiency

Active Publication Date: 2020-08-28
ZHENGZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, there is no report on the use of Sumo-labeled E. coli system to promote the expression of HPV 16 L1 and to prepare cervical cancer vaccines.

Method used

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  • Recombinant plasmid expressing soluble human papillomavirus 16 subtype l1 protein and its expression method
  • Recombinant plasmid expressing soluble human papillomavirus 16 subtype l1 protein and its expression method
  • Recombinant plasmid expressing soluble human papillomavirus 16 subtype l1 protein and its expression method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The construction of the recombinant expression plasmid of embodiment 1 band SUMO tag

[0065] The recombinant expression plasmid with the SUMO tag of the present invention is rebuilt on the basis of the pET28a backbone, and the specific steps are as follows:

[0066] First, introduce the restriction endonuclease Nco I restriction endonuclease Nco I digestion site and 6*His-tag gene at the upstream 5' end of the SUMO gene, and introduce the restriction endonuclease Bsa I restriction endonuclease Bsa I restriction endonuclease restriction site at the downstream 3' end, using Sumo The special properties of the protease cleavage site (the cleavage site is Gly Gly, which encodes the gene GG AGGT ), combined with the specificity of the Bsa I restriction site, can realize the N-terminal non-residual cleavage of the recombinantly expressed protein.

[0067] The Bsa I restriction site is:

[0068] The sequence of the Sumo-tag tag gene is as follows:

[0069] CCATGGGTCATCAC...

Embodiment 2

[0092] Example 2 Cloning of HPV16 L1 gene

[0093] 1. Design and synthesis of HPV16 L1 gene sequence

[0094] The HPV16 L1 gene sequence of the present invention is a DNA sequence optimized by Escherichia coli preferred codons, that is, a DNA sequence obtained by codon optimization and certain corrections to the optimal codon frequency, specifically as follows:

[0095] Firstly, the natural HPV 16L1 gene is modified, and all its amino acids use the most frequently used codons to design a brand new HPV16 L1 DNA sequence. At the same time, in order to avoid the GC ratio of the translated mRNA being too high, The secondary structure affects the translation efficiency and avoids some commonly used enzyme cutting sites. It is necessary to make some corrections to the optimal codon frequency, and finally obtain a brand new HPV16 L1 DNA sequence, as follows:

[0096] ATGTCTCTGTGGCTGCCTTCTGAGGCCACTGTCTACCTGCCTCCTGTCCCAGTATCTAAGGTTGTAAGCACCGATGAATATGTTGCACGCACCAACATCTATTATCATGCAGGTACC...

Embodiment 3

[0109] Example 3 Construction of recombinant plasmid expressing soluble human papillomavirus 16 subtype L1 protein

[0110] The recombinant plasmid pET28-Sumo and the PCR amplification product (HPV16 L1 gene) were subjected to double digestion with restriction endonucleases Bsa I and Xho I respectively, and the specific reaction conditions were as follows:

[0111] The enzymatic digestion system is:

[0112]

[0113] The total volume of the system is 50 μL, and the reaction conditions are 37° C. for 4 hours.

[0114] Subsequently, the double restriction products of the recombinant plasmid pET28-Sumo and the HPV16 L1 gene were respectively recovered and ligated with T4 DNA ligase to obtain the recombinant plasmid II. The ligation system was as follows:

[0115]

[0116] The total reaction system is 20 μl, and the specific reaction conditions are: overnight connection at 16°C.

[0117] The recombinant plasmid II was transformed into Escherichia coli competent cells JM109...

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Abstract

The invention discloses a recombinant plasmid capable of expressing a soluble human papilloma virus 16 subtype L1 protein and an expression method thereof. The recombinant plasmid is constructed by inserting a human papilloma virus 16 subtype L1 gene and an SUMO label gene into an expression plasmid. According to the recombinant plasmid disclosed by the invention, the HPV16 L1 gene is optimized, so that the HPV16 L1 gene is more applicable to efficient expression of a target protein in an escherichia coli host; meanwhile, an Sumo label expression system is initially applied to fusion expression in escherichia coli, and the solubility of the target protein is further enhanced, so that the expressed target protein has higher activity. The expression method disclosed by the invention has the advantages that the target protein solubility expression efficiency is obviously improved, the product property is uniform and stable, the target protein expression quantity in a supernatant of a fermented disrupted bacterium liquid can reach 70Mug / ml, and the numerical value is obviously higher than yields of the target protein in other prokaryotic expression systems, so that the industrial production requirement is met.

Description

technical field [0001] The invention relates to a recombinant plasmid expressing soluble human papillomavirus 16 subtype L1 protein, a construction method, a recombinant engineering bacterium and an expression method thereof, and belongs to the technical field of genetic engineering. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) is a kind of double-stranded small molecule DNA virus with strict species specificity. It mainly infects human skin and mucous membrane tissues, causing proliferative lesions of epithelial tissues in corresponding parts. In 1977, Laverty (Laverty, C.R., Russell, P.et al.1977) observed the presence of HPV particles in cervical cancer tissue in electron microscopy. In 1981, Zur Hausen (H zur Hausen.et al.1981) proposed that HPV may be related to cervical cancer. Hypotheses related to carcinogenesis. Since then, this view has been confirmed by more and more experiments. The incidence of cervical cancer is directly rela...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/37C12N15/66C12N1/21A61K39/12A61P31/20C12R1/19
Inventor 王爱萍张改平陈玉梅王娟刘运超周景明祁艳华刘东民刘燕凯刘文英栗宁蒋敏
Owner ZHENGZHOU UNIV
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