Unlock instant, AI-driven research and patent intelligence for your innovation.

Gene sequence of an optimized l1 protein of human papillomavirus type 31

A technology of human papillomavirus and L1 protein, which is applied in the field of molecular virology and immunology, and can solve the problems of high price, difficult target protein, and low expression level

Active Publication Date: 2017-04-12
XIAMEN UNIV +1
View PDF2 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Gene sequence of an optimized l1 protein of human papillomavirus type 31
  • Gene sequence of an optimized l1 protein of human papillomavirus type 31
  • Gene sequence of an optimized l1 protein of human papillomavirus type 31

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

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

[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 a...

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] Massive Expression of HPV31 Ctag-L1 Protein

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

[0093] The 50L fermenter produced by Shanghai Baoxing Biological Company was used for large-scale cultivation. Calibrate the pH electrode of the fermenter, put 30L LB medium into the fermenter, and sterilize at 121°C for 30 minutes in situ; calibrate the dissolved oxygen electrode, set the zero po...

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 the electrophoresis, the HPV31L1-specific antibodies 8G6 and 11D2 were used for Western detection. The results are shown in image 3 . The results showed that both the 55kD target band and the 60kD heteroprotein band could react with HPV31L1-specific antibodies 8G6 and 11D2, which proved that the band contained HPV31L1 protein.

[0134] Mass spectrometric analysis of 60kD protein bands of HPV31Ctaa-L1 and HPV31Ctga-L1

[0135] The HPV31taa-L1 protein and the HPV31Ctga-L1 protein were subjected to electrophoresis respectively, and after the electrophoresis was completed, stained with Coomassie Brilliant Blue, and the 60kD miscellaneous protein band above the 55kD target band was cut off. Protein gel pieces were decolorized twice with 40 μL decolorizing sol...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
diameteraaaaaaaaaa
molecular weightaaaaaaaaaa
radiusaaaaaaaaaa
Login to View More

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 whose stop codon is TAG, its encoded protein and preparation method, and virus-like particle (Virus-Like Particle) comprising its encoded protein , VLP), the protein 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 protein and virus-like particles in the preparation of a pharmaceutical composition or vaccine, which is used to prevent 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 vi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(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