HPV58 L1 gene, vector, strain and expression method

A gene and strain technology, applied in chemical instruments and methods, microbial-based methods, botanical equipment and methods, etc., can solve the problems of low expression level and no expression, achieve high expression level, high expression level, and benefit large-scale The effect of large-scale industrial production

Active Publication Date: 2010-10-13
SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in expression systems such as Escherichia coli, Pichia pastoris, and baculovirus, L1 protein is often limited by the frequency of amino acid codon usage in these organisms, resulting in low or no expression.

Method used

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  • HPV58 L1 gene, vector, strain and expression method
  • HPV58 L1 gene, vector, strain and expression method
  • HPV58 L1 gene, vector, strain and expression method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: HPV58 L1 codon optimization design

[0041] According to the wild-type HPV58 L1 amino acid sequence (GenBank: D90400.1, SEQ ID NO: 2, figure 1 ) to obtain the wild-type 58L1 nucleotide sequence. The wild-type HPV 58L1 DNA sequence was modified, and all codons used in Pichia pastoris with high or highest frequency were used, and the formation of secondary structure and the selection of enzyme cutting sites were considered, and finally the HPV58 of the present invention was obtained Nucleotide sequence (SEQ ID NO: 1, figure 2 ).

Embodiment 2

[0042] Embodiment 2: Construction of HPV58 L1 recombinant expression vector

[0043] Synthesize the HPV58 L1 gene sequence of the present invention shown in SEQ ID NO: 1 and carry out PCR amplification as the template of PCR amplification, and it is cloned into pPICZαB vector (the described vector is purchased from Invitrogen, catalog number: V195-20).

[0044] Firstly, the 58L1 DNA fragment with BstBI and KpnI at both ends was amplified by PCR.

[0045] PCR primers:

[0046] Forward primer: 5'-ACTAATTA TTCGAA ACGATGTCTGTTTGGAGAC-3' (the underlined sequence is the BstBI restriction site);

[0047] Reverse primer: 5'-AGC GGTACC CTATTACTTCTTAACC-3' (the underlined sequence is the KpnI restriction site).

[0048] PCR reaction system:

[0049] Reactant

Volume (μL)

wxya 2 o

40.5

Forward primer (20pmol)

1

Reverse primer (20pmol)

1

10*buffer (with Mg2+)

5

dNTP (10mM each, Takara)

1.5

DNA t...

Embodiment 3

[0052] Example 3: Construction and expression of HPV58 L1 recombinant expression strain

[0053] The pPICZ58L1 vector was linearized with Sad. After the digestion reaction, the protein was removed with a phenol:chloroform (1:1) solution, and then 2.5 times the volume of absolute ethanol and 1 / 10 volume of 3M NaAc (pH 5.2) solution were added to precipitate the DNA. The resulting precipitate was washed with 75% ethanol, dried and washed with a small amount of sterile ddH 2 O dissolved the precipitate, electroporated into Pichia pastoris X-33 strain (Invitrogen), and spread on YPDS plate (1% Trypton, 2% Peptone, 2% glucose, 1M sorbitol) (containing 200 μg / ml Zeocin), 30 Cultivate for 3 days to obtain hundreds of clones. Dozens of clones were picked and inoculated on YPD plates (1% Trypton, 2% Peptone, 2% glucose, 1.5% agar powder) (containing 1500 μg / ml Zeocin) for screening high-copy plasmid strains, and cultured at 30°C for 2 days. Some clones grew faster, and several clone...

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Abstract

The invention relates to a gene of the main capsid protein of 58 type human papillomavirus, which is optimized through a pichiapastoris expression codon, a vector containing the gene, a strain and an expression method thereof.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular, the present invention relates to the gene of the main capsid protein L1 of type 58 human papillomavirus suitable for expression in Pichia pastoris through codon optimization, and the vector containing the gene, Bacterial strains and methods for expressing the gene. Background technique [0002] Cervical cancer is the second most common gynecological malignancy after breast cancer. More than 500,000 women worldwide are diagnosed with cervical cancer every year, and 270,000 women die from the disease, with an age-standardized infection rate of 10.5%. As early as the early 1980s, Harald zur Hausen discovered that human papillomavirus (HPV) infection was related to the incidence of cervical cancer, and a large number of subsequent studies have also proved that HPV is closely related to cervical cancer and its precancerous lesions. So far, more than 100 HPV genotypes have been ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/37C12N15/81C12N1/19C07K14/025C12R1/84
Inventor 沈琼雷建强傅文彬张梦华
Owner SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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