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Optimized gene sequence of human papillomavirus 31 type L1 protein

A technology of human papillomavirus and L1 protein, which is applied in the field of molecular virology and immunology, and can solve problems such as difficult to apply in large-scale production, many types of miscellaneous proteins, and difficult target proteins

Active Publication Date: 2014-12-17
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the HPV L1 proteins expressed by E. coli lose their natural conformation and cannot produce protective antibodies against HPV.
Or although the above protein can be purified by inclusion bodies, renaturation and other steps can also obtain HPV VLP (Kelsall, S.R. and J.K.Kulski(1995).J Virol Methods53(1):75-90), but the amount of protein loss in the renaturation process Large, low yield, therefore, difficult to apply in large-scale production
Although HPV L1 protein can also be solublely expressed in the correct conformation in Escherichia coli and dissolved in the lysed supernatant of the bacteria, its expression level is low, and there are many types and large amounts of foreign proteins in the supernatant, and it needs to be purified from it. The target protein is quite difficult
Although there are also reports in the literature that the expression of L1 protein in the supernatant can be increased by means of GST fusion expression, and it can help the purification of the protein of interest (Li, M., T.P.Cripe, et al. (1997). J Virol71 (4): 2988-95), but the cleavage of fusion proteins often requires expensive enzymes, which still cannot be applied to large-scale production

Method used

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  • Optimized gene sequence of human papillomavirus 31 type L1 protein
  • Optimized gene sequence of human papillomavirus 31 type L1 protein
  • Optimized gene sequence of human papillomavirus 31 type L1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1. Construction of expression plasmids encoding HPV31L1 gene with different stop codons

[0080] Preparation of full-length HPV31L1 gene fragment used as template

[0081] The full-length HPV31L1 gene fragment used as template was synthesized by Shanghai Boya Company. The synthesized gene was optimized according to the coding sequence of HPV31L1 with the sequence number J04353.1 in the NCBI GenBank database, and was optimized according to the codon bias of Escherichia coli. The full length of the fragment was 1515bp, and the stop codon was TAA (SEQ ID NO: ID NO.2). On the basis of this artificially synthesized full-length HPV31L1 gene fragment, the polynucleotide sequence encoding HPV31L1 of the present invention is prepared on the basis of HPV31.

[0082] Construction of non-fusion expression vector encoding HPV31L1 gene with different stop codons

[0083] The HPV31L1 full-length gene fragment obtained in the previous step with TAA as the stop codon was used...

Embodiment 2

[0089] Example 2. Expression and purification of HPV31L1 protein encoded by genes with different stop codons

[0090] This paper takes HPV31Ctag-L1 as an example to describe the expression and purification of HPV31L1 protein.

[0091] Mass expression of HPV31Ctag-L1 protein

[0092] Take out the Escherichia coli strain carrying the recombinant plasmid pTO-T7-HPV31Ctag-L1 from -70°C, inoculate it into 50ml LB liquid medium containing kanamycin, and cultivate at 200rpm and 37°C for about 8 hours; then transfer to Inoculated into 10 bottles of 500ml LB medium containing kanamycin (5ml bacterial solution per bottle), cultivated overnight at 200rpm and 37°C as seed solution.

[0093] Large-scale culture was carried out in a 50L fermenter produced by Shanghai Baoxing Biological Company. Correct the pH electrode of the fermenter, put 30L of LB medium into the fermenter, and sterilize it in-situ at 121°C for 30 minutes; calibrate the dissolved oxygen electrode, set the zero point af...

Embodiment 3

[0131] Example 3. Identification and analysis of HPV31taa-L1 protein and HPV31tga-L1 protein

[0132] Western blot detection of HPV31Ctaa-L1

[0133] The purified HPV31taa-L1 protein was subjected to electrophoresis. After electrophoresis, the HPV31L1-specific antibodies 8G6 and 11D2 were used for Western detection respectively. The results are shown in image 3 . The results showed that both the 55kD target band and the 60kD hybrid protein band could react with HPV31L1 specific antibodies 8G6 and 11D2, which proved that the band contained HPV31L1 protein.

[0134] Mass Spectrometry Analysis of 60kD Protein Bands of HPV31Ctaa-L1 and HPV31Ctga-L1

[0135] HPV31taa-L1 protein and HPV31Ctga-L1 protein were electrophoresed respectively. After electrophoresis, Coomassie brilliant blue staining was used to cut out the 60kD heteroprotein band above the 55kD target band. The protein gel block was decolorized twice with 40 μL of decolorizing solution (30% ACN, 50 mmol / L ammonium ace...

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Abstract

The invention relates to the field of molecular virology and immunology, and particularly, relates to a gene sequence for encoding a human papillomavirus 31 type L1 protein and with a termination codon of TAG, and an encoded protein and a preparation method thereof, and virus-like particles containing the encoded protein; the protein and the virus-like particles can be used for preventing HPV (especially HPV31) infection and diseases such as cervical cancer and the like caused by the HPV (especially HPV31) infection. The invention also relates to an application of the protein and the virus-like particles in preparation of a pharmaceutical composition or a vaccine. The pharmaceutical composition or the vaccine is used for preventing the HPV (especially HPV31) infection and the diseases such as cervical cancer and the like caused by the HPV (especially HPV31) infection.

Description

technical field [0001] The present invention relates to the fields of molecular virology and immunology. Specifically, the present invention relates to a gene sequence encoding human papillomavirus type 31 L1 protein with a stop codon of TAG, the encoded protein and a preparation method, and a virus-like particle (Virus-Like Particle) comprising the encoded protein , VLP), the proteins and virus-like particles can be used to prevent HPV (especially HPV31) infection and diseases caused by HPV (especially HPV31) infection such as cervical cancer. The present invention also relates to the use of the above-mentioned proteins and virus-like particles for the preparation of pharmaceutical compositions or vaccines for preventing HPV (especially HPV31) infection and HPV (especially HPV31) infection caused by Diseases such as cervical cancer, etc. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) is a non-enveloped DNA virus. The diameter of the virus ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/37C12N15/70C12N1/21C12N7/04A61K39/12A61K48/00A61P31/20A61P35/00C12R1/19
Inventor 李少伟魏旻希范飞王大宁潘晖榕张军夏宁邵
Owner XIAMEN UNIV
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