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Method for producing HPV31 L1 protein by using hansenula polymorpha expression system

A Hansenula protein technology, applied in the field of Hansenula expression system to produce HPV31L1 protein, can solve the problems that the purity of exogenous protein is not disclosed, and the protein expression level is not provided.

Pending Publication Date: 2019-12-20
BEIJING ABZYMO BIOSCIENCES CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of the induced expression of foreign proteins, the induction time of HPV16 and HPV18 L1 in the fermenter needs to be more than 20 hours, and no specific information on the protein expression level is provided
In addition, as an important indicator of protein purification, there is no disclosure about the purification process and the purity of the exogenous proteins HPV16 and HPV18 L1 when the purification is completed

Method used

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  • Method for producing HPV31 L1 protein by using hansenula polymorpha expression system
  • Method for producing HPV31 L1 protein by using hansenula polymorpha expression system
  • Method for producing HPV31 L1 protein by using hansenula polymorpha expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Analysis of HPV31 L1 Consensus Amino Acid Sequence

[0055] The full-length HPV31 L1 protein consists of 504 amino acids. After searching GenBank, use the AlignX function of the Vector NTI software to perform amino acid sequence alignment and analysis, and obtain the most representative HPV31 L1 consensus amino acid sequence (consensus amino acid sequence, that is, in HPV31 Each amino acid position of L1 adopts the sequence of the amino acid residue with the highest frequency), and its sequence is shown in SEQ ID NO:1.

Embodiment 2

[0056] Example 2: Optimal Design and Artificial Synthesis of HPV31 L1 Encoding Gene

[0057] In order to efficiently express the HPV31 L1 protein using Hansenula, the inventors optimized the codons of the nucleotide sequence for Hansenula according to the amino acid sequence shown in SEQ ID NO:1. The optimization principles include: a) select codons with the highest or higher frequency of use according to the Hansenula genetic code usage frequency table; b) avoid negative regulatory elements that have potential effects on gene transcription or protein translation, such as PolyAT regions, PolyGC regions, Silencer region and internal splicing sites, etc.; c) Comprehensive analysis of the mRNA secondary structure including the 5' end UTR, HPV31L1 coding region and 3' end UTR, to avoid the formation of complex RNA secondary structures, Reduce the free energy of the secondary structure of the mRNA; d) Use the 5'UTR region that is completely consistent with the natural sequence down...

Embodiment 3

[0059] Example 3: Generation of expression constructs carrying the HPV31 L1 nucleotide sequence

[0060] The Hansenula expression vector used in the present invention is the Hansenula expression vector pRMHP2.1 (SEQ ID NO: 9) described in the Chinese patent application with application number 201210021524.X.

[0061] (1) PCR amplification of MOX promoter and MOX terminator

[0062] Using the mixed genomic DNA of Hansenula strains ATCC26012 and ATCC34438 as a template, the following primers were used to amplify the MOX promoter with a size of 1518bp, and a NotI restriction site was introduced upstream;

[0063] MOX promoter upstream primer: 5'-AAGGAAAAAAGCGGCCGCAACGATCTCCTCGAGCTGCTCGC-3' (SEQ ID NO: 3)

[0064] MOX promoter downstream primer: 5'-TTTGTTTTTGTACTTTAGATTGATGTC-3' (SEQ ID NO: 4)

[0065] Using the mixed genomic DNA of Hansenula strains ATCC26012 and ATCC34438 as a template, the following primers were used to amplify the MOX terminator with a size of 311bp, and at ...

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Abstract

The invention relates to a method for producing HPV31 L1 protein by using a hansenula polymorpha expression system. Specifically, the invention discloses a method for producing recombinant hansenula polymorpha cells expressing the HPV31 L1 protein and the recombinant hansenula polymorpha cells produced by the method. The invention also discloses a method for producing the HPV31 L1 protein by usingthe recombinant hansenula polymorpha cells and application of the produced HPV31 L1 protein in preparation of a preventive vaccine.

Description

[0001] This application is a divisional application of an invention patent application with an application date of May 17, 2013, an application number of 201310185027.8, and an invention title of "Method for Producing HPV31 L1 Protein Using Hansenula Yeast Expression System". [0002] field of invention [0003] The invention belongs to the technical field of medical bioengineering, and relates to a method for producing HPV31 L1 protein, in particular to a method for producing HPV31 L1 protein with a Hansenula expression system. Background technique [0004] Human papillomavirus (human papillomavirus, HPV) is a non-enveloped closed-circle double-stranded DNA virus, belonging to the Papovaviridae polyomavirus subfamily, which mainly invades human epithelial mucosal tissues, and then induces various benign and malignant diseases. Proliferative lesions. [0005] At present, more than 200 different subtypes of HPV have been identified. HPV infection has obvious tissue specificity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12P21/02C12N1/19C07K14/025C07K1/16A61K39/12A61P31/20
Inventor 霍烛于跃程海刘娟王贻杰陈丹李鼎锋刘勇
Owner BEIJING ABZYMO BIOSCIENCES CO LTD
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