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Preparation method of human papillomavirus and heat shock protein recombinant protein

A technology of human papillomavirus and heat shock protein, which is applied in the field of bioengineering, can solve problems such as the difficulty of expressing and purifying HPVE6, the inability to guarantee safety, and the impact on the approval of new drugs, etc., to achieve improved renaturation rate and purity, low cost, and easy operation convenient effect

Active Publication Date: 2021-02-02
北京云禾天秤生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, it is very difficult to express and purify HPV E6 from Escherichia coli (E.coli) (Two-stepchromatographic purification of glutathione S-transferase-tagged humanpapillomavirus type 16E6protein and its application for serology, ProteinExpression and Purification Volume 132, April 2017, Pages 19-26), because of its strong hydrophobicity, it is easy to produce protein aggregates, which affects subsequent purification
Therefore, when purifying HPV E6-related recombinant proteins, the research data reported at home and abroad are to design purification tags (such as GST, MBP, HIS, etc.) on related recombinant proteins, and use affinity chromatography to purify to obtain high-purity target proteins. For example, the E. coli expression system expresses human papillomavirus (HPV) type 16 E6E7HSP65 fusion protein using HIS tag purification, and the redundant tag (redundant amino acid) of the final target protein is not removed, although the redundant amino acid cannot be seen in the short term Side effects, but the safety of long-term use cannot be guaranteed, and it will affect the approval of new drugs

Method used

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  • Preparation method of human papillomavirus and heat shock protein recombinant protein
  • Preparation method of human papillomavirus and heat shock protein recombinant protein
  • Preparation method of human papillomavirus and heat shock protein recombinant protein

Examples

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Embodiment 1

[0050] Synthesis of the optimized codon gene of embodiment 1 human papillomavirus (HPV) 16 type E6E7HSP70 fusion protein

[0051] According to the obtained HPV16 early gene E6 and E7 encoded polypeptide amino acid sequence and mycobacterial heat shock protein 70 gene, adopt HPV16 early gene E6, E7 and mycobacterium heat shock protein 70 protein sequence combination design as shown in SEQID NO:2 indicated fusion protein.

[0052] Utilizing the degeneracy and preference of the gene, the optimized codon nucleotide sequence was designed under the premise that the amino acid sequence of the encoded product protein remained unchanged. The inventor named the sequence E6E7HSP70. Experimental testing proved that the optimized gene It is suitable for expressing E6E7HSP70 fusion protein in Escherichia coli, and the optimized gene sequence is shown in SEQ ID NO:1.

Embodiment 2

[0053] Embodiment 2 Construction of prokaryotic expression recombinant plasmid

[0054] The synthesized and cloned E6E7HSP70 optimized gene was digested with restriction enzymes, inserted between the NcoI and BamH I restriction sites of the Escherichia coli expression vector pET28a, identified by NcoI and BamH I restriction and sequenced and screened to obtain the correct inserted prokaryotic Expression of recombinant plasmid pET28a-E6E7HSP70.

Embodiment 3

[0055] The construction of embodiment 3 escherichia coli engineering bacteria (recombinant bacteria)

[0056] The recombinant plasmid pET28a-E6E7HSP70 obtained in Example 2 was used to transform BL21(DE3) Escherichia coli, and cultured overnight at 37° C. after spreading on a plate. Picking a single spot in LB medium (see references for the medium recipe: Sambrook J, Fritsch EF, and Manny Artis T, Molecular Cloning Experiment Guide. 1992. Second Edition (Beijing): Science Press : page 908), when OD 600 = 0.6, adding IPTG with a final concentration of 0.4mM to induce expression for 2, 3, and 4 hours respectively, and taking an appropriate amount of bacteria for SDS-PAGE gel electrophoresis analysis, the results are shown in figure 1 , there is a newly added protein band at the molecular weight of about 49KD, this protein band (recombinant protein) accounts for about 20% of the total protein output of the whole bacteria, and the positive clone is preserved as the original bacte...

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Abstract

The present invention provides a preparation method of human papillomavirus and heat shock protein recombinant protein, comprising: A, constructing engineering bacteria expressing human papillomavirus and heat shock protein recombinant protein; B, inducing expression and purifying target protein; wherein, The engineering bacterium is obtained by constructing the coding gene of the recombinant protein on the pET28a plasmid after codon optimization, and transforming it into Escherichia coli. The optimized genes of human papillomavirus and heat shock protein recombinant protein provided by the present invention can be highly expressed, and the expression level of the target protein accounts for 20% of the total bacterial protein. In the process of protein purification, inclusion bodies are dissolved with sodium lauryl sarcosine, combined with on-column renaturation, which effectively avoids complex processes, reduces the use of denaturant urea or guanidine hydrochloride, and reduces waste liquid discharge. It has many advantages such as low cost, high renaturation rate and convenient operation, and provides a new idea for the preparation of human papillomavirus and heat shock protein recombinant protein.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a preparation method of human papilloma virus and heat shock protein recombinant protein. Background technique [0002] Among gynecological tumors, the incidence of cervical cancer is second only to breast cancer, ranking second. About 200,000 women worldwide die from this disease every year. Statistics in recent years show that there are about 131,500 new cervical cancer patients in my country every year, accounting for 28% of the total number of new cervical cancer cases in the world. About 290,000 women die of cervical cancer every year in the world, of which about 50,000 are in my country. , The rising trend of young cervical cancer patients is obvious. According to reports, the occurrence of cervical cancer is closely related to human papillomavirus (HPV) infection, and some high-risk HPV virus infections are likely to eventually lead to cervical cancer. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C12R1/19
CPCC07K14/00C07K14/005C12N15/70C12N2800/22
Inventor 孙昌荣李利华李慎涛王志
Owner 北京云禾天秤生物科技有限公司
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