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Method using hansenula expression system for production of HPV52 L1 protein

A Hansenula, protein technology, applied in the field of Hansenula expression system to produce HPV52L1 protein, can solve the problem that the purity of exogenous protein is not disclosed, and the protein expression level is not provided.

Active Publication Date: 2014-10-29
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 HPV18L1 in the fermenter needs to exceed 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 HPV18L1 when the purification is completed

Method used

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  • Method using hansenula expression system for production of HPV52 L1 protein
  • Method using hansenula expression system for production of HPV52 L1 protein
  • Method using hansenula expression system for production of HPV52 L1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Analysis of HPV52L1 Consensus Amino Acid Sequence

[0052] The full-length HPV52L1 protein consists of 503 amino acids. After being searched by GenBank, use the AlignX function of the Vector NTI software for amino acid sequence alignment analysis to obtain the most representative HPV52L1 consensus amino acid sequence (consensus amino acid sequence, that is, in each HPV52L1 Amino acid positions all adopt the sequence of amino acid residues with the highest frequency of occurrence), the sequence of which is shown in SEQ ID NO:1.

Embodiment 2

[0053] Example 2: Optimal Design and Artificial Synthesis of HPV52L1 Encoding Gene

[0054] In order to efficiently express the HPV52L1 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 region, PolyGC region, Silencer region and internal splicing sites, etc.; c) Comprehensive analysis of mRNA secondary structure including 5' end UTR, HPV52L1 coding region and 3' end UTR, to avoid the formation of complex RNA secondary structure, Reduce the free energy of the secondary structure of mRNA; d) Use the 5'UTR region that is completely consistent with the natural sequence downstream of the Han...

Embodiment 3

[0056] Example 3: Generation of expression constructs carrying the HPV52L1 nucleotide sequence

[0057] 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.

[0058] (1) PCR amplification of MOX promoter and MOX terminator

[0059] 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;

[0060] MOX promoter upstream primer: 5'-AAGGAAAAAAGCGGCCGCAACGATCTCCTCGAGCTGCTCGC-3' (SEQ ID NO: 3)

[0061] MOX promoter downstream primer: 5'-TTTGTTTTTGTACTTTAGATTGATGTC-3' (SEQ ID NO: 4)

[0062] 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 t...

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Abstract

The invention relates to a method using a hansenula expression system for production of HPV52 L1 protein, and specifically discloses a method for production of recombinant hansenula cells for expression of the HPV52 L1 protein, and the recombinant hansenula cells produced by the method. The invention also discloses a method using the recombinant hansenula cells to produce the HPV52L1 protein and use of the HPV52L1 protein produced by the method in the preparation of preventive vaccines.

Description

field of invention [0001] The invention belongs to the technical field of medical bioengineering and relates to a method for producing HPV52L1 protein, in particular to a method for producing HPV52L1 protein with a Hansenula expression system. Background technique [0002] 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. [0003] At present, more than 200 different subtypes of HPV have been identified. HPV infection has obvious tissue specificity. Different types of HPV have different tropisms for skin and mucous membranes, and can induce different papillary lesions. There are about 30 types. HPV types are associated with reproductive tract infections, and more than 20 of them are associated with tumors. According to the differe...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/37C12N15/81C12P21/02A61K39/12A61P31/20C12R1/78
Inventor 于跃班靖洋霍烛陈丹王贻杰刘娟程海李鼎锋刘勇
Owner BEIJING ABZYMO BIOSCIENCES CO LTD
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