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16 type human papilloma virus major capsid protein L1 gene

A technology of papilloma virus and its main capsid protein, which is applied in the field of bioengineering to achieve the effects of low cost, high yield, and uniform and stable product properties

Active Publication Date: 2009-05-27
SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] At present, there is no report of using the Pichia pastoris expression system to express HPV16L1 and 18L1 for the preparation of cervical cancer vaccines. The present invention uses the Pichia pastoris system to prepare 16L1 and 18L1 for the first time, and proves that VLP can be formed in cells, and after fermentation and purification, Purified samples can make mice produce higher titers of antibodies, and prove that they have neutralizing activity through a virus neutralization model

Method used

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  • 16 type human papilloma virus major capsid protein L1 gene
  • 16 type human papilloma virus major capsid protein L1 gene
  • 16 type human papilloma virus major capsid protein L1 gene

Examples

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Embodiment 1

[0032] Example 1 , Design and synthesis of codon-optimized L1 gene of HPV16

[0033] 1.1. Design of codon-optimized L1 gene of HPV16

[0034] The present invention relates to a DNA molecule encoding the main capsid protein L1 of human papillomavirus type 16 (HPV16) optimized by the preferred codon of Pichia pastoris. The three segments are obtained by optimizing the codon and correcting the frequency of the optimal codon. DNA sequence, its sequence is shown in SEQ ID NO: 1, 2 and 3 respectively, specifically as follows:

[0035] Firstly, the natural HPV16L1 gene is modified, and all amino acids of it are codons with the highest frequency of use, and a brand-new HPV DNA sequence, namely SEQ ID NO:1, is designed.

[0036] Then, in order to avoid the GC ratio of the translated mRNA being too high, the influence of the secondary structure of the mRNA on the translation efficiency, and to avoid some commonly used enzyme cutting sites, some corrections are made to the optimal cod...

Embodiment 2

[0052] Example 2 , Expression vector construction of HPV 16L1 gene

[0053] Cloning the optimized sequence of SEQ ID NO: 1 into the expression vector of Pichia pastoris, the construction method is as follows figure 1 As shown, the specific steps are as follows:

[0054] 2.1. Synthesize the forward primer and reverse primer required to amplify HPV 16L1, the sequence is as follows:

[0055] Forward primer: 5'-ACTAATTA TTCGAA ACGATGTCTTTTGTGG-3';

[0056] Reverse primer: 5'-AGC GGTACC CTATTACAACTTTTCTCTTTCTTTC-3'.

[0057] Wherein, the forward primer includes a BstBI restriction endonuclease site; the reverse primer includes a KpnI restriction endonuclease site positioned at the flank of the stop codon, and the enzyme cutting sites are respectively shown in the underline in the primer sequence .

[0058] 2.2. Using the above primer pair as primers, and using the pUC-16L1 obtained in Example 1 as a template, PCR amplification was performed, and the amplified product was ...

Embodiment 3

[0060] Example 3 , Construction and expression of HPV 16L1 Pichia pastoris expression strain

[0061] 3.1. Construction of HPV 16L1 Pichia pastoris expression strain

[0062] The pPICZ-16L1 plasmid was single-digested with Sac I restriction endonuclease to make it linearized, and the empty plasmid pPICZaB was subjected to the same digestion as a negative control.

[0063] Add absolute ethanol to the enzyme digestion reaction solution to obtain DNA precipitation, dissolve the linearized pPICZ-16L1 fragment with a small amount of double distilled water, and transform Pichia pastoris X-33 (Invitrogen) by electroporation. The electroporation conditions are:

[0064] The DNA fragment is 5 micrograms, the voltage is 1500 volts, the resistance is 25 ohms, and the electric shock time is 5 milliseconds.

[0065] The electroporation products were plated on YPDS agar containing 200 μg / ml Zeocin. Since the pPICZaB vector carried the Zeocin resistance gene, the transformation products c...

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Abstract

The invention provides an L1 major capsid protein gene of human papillomavirus type 16 and a pichia pastoris strain expressing the gene. The gene sequence of the L1 protein is the gene sequence which is optimized by codon preferred by pichia pastoris, and corrects the optimized codon frequency to express the L1 protein. The L1 major capsid protein gene of human papillomavirus type 16 is an optimized L1 gene, and has the advantages that the optimized gene is more suitable for high expressing target proteins in pichia pastoris host and can meet the requirement of industrial production; meanwhile, a pichia pastoris expression system is adopted, so the cost is low, the yield is high, and the product nature is more uniform and stable.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a human papillomavirus capsid protein gene, more specifically to a type 16 human papillomavirus (HPV16) main capsid protein L1 gene. Background technique [0002] About 500,000 women in the world suffer from cervical cancer every year (Koutsky LA.et al.Epidemiol Rev 1988:10:122-163), of which about 270,000 die from cervical cancer (more than 80% in developing countries). second only to breast cancer. [0003] In 1977, Laverty (Laverty CR.et al.Acta Cytol.1977; 21:114-117) observed the presence of HPV particles in cervical cancer tissue under electron microscopy. In 1981, Zur Hausen (H zur Hausen.et al.Gynecol Oncol.1981 :12:s124-128) put forward the hypothesis that HPV may be related to the pathogenesis of cervical cancer. Since then, this view has been confirmed by more and more experiments. The incidence of cervical cancer is directly related to human papilloma virus (HPV) infe...

Claims

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Application Information

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IPC IPC(8): C12N15/37C12N15/81C12N1/19C12R1/84
Inventor 张高峡沈琼雷建强袁靖宇张千里魏健吴克
Owner SHANGHAI ZERUN BIOTECHNOLOGY CO LTD
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