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Method for generation of HPV58 L1 protein by Hansenula expression system

A Hansenula protein technology, applied in the field of Hansenula expression system to produce HPV58L1 protein, can solve the problems 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 for generation of HPV58 L1 protein by Hansenula expression system
  • Method for generation of HPV58 L1 protein by Hansenula expression system
  • Method for generation of HPV58 L1 protein by Hansenula expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Analysis of HPV58L1 Consensus Amino Acid Sequence

[0052] The full-length HPV58L1 protein consists of 498 amino acids. After searching GenBank, use the AlignX function of the Vector NTI software for amino acid sequence alignment analysis to obtain the most representative HPV58L1 consensus amino acid sequence (consensus amino acid sequence, that is, each HPV58L1 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 HPV58L1 Encoding Gene

[0054] In order to efficiently express the HPV58L1 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, HPV58L1 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 HPV58L1 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 for generation of HPV58 L1 (human papillomavirus) protein by a Hansenula expression system. Specifically, the invention discloses a method for generation of a recombinant Hansenula cell for expressing the HPV58L1 protein and the recombinant Hansenula cell generated thereby. The invention also discloses a method for generating the HPV58L1 protein by the recombinant Hansenula cell and application of the generated HPV58L1 protein in 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 HPV58L1 protein, in particular to a method for producing HPV58L1 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 differ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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