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Method for producing HPV45 L1 protein by using Hansenula polymorpha expression system

A technology of Hansenula yeast and protein is applied in the field of Hansenula yeast expression system to produce HPV45L1 protein, which can solve the problems of not providing protein expression level and undisclosed purity of exogenous protein.

Active Publication Date: 2014-11-26
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 producing HPV45 L1 protein by using Hansenula polymorpha expression system
  • Method for producing HPV45 L1 protein by using Hansenula polymorpha expression system
  • Method for producing HPV45 L1 protein by using Hansenula polymorpha expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Analysis of HPV45L1 consensus amino acid sequence

[0051] The full-length HPV45L1 protein consists of 510 amino acids. After the GenBank search, the AlignX function of the Vector NTI software was used for amino acid sequence alignment analysis to obtain the most representative HPV45L1 consensus amino acid sequence (consensus amino acid sequence, that is, in each HPV45L1 amino acid sequence). The amino acid positions are all using the sequence of the amino acid residue with the highest frequency), and its sequence is shown in SEQ ID NO: 1.

Embodiment 2

[0052] Example 2: Optimization design and artificial synthesis of HPV45L1 encoding gene

[0053] In order to efficiently express HPV45L1 protein with Hansenula, the inventors performed codon optimization of the nucleotide sequence for Hansenula according to the amino acid sequence shown in SEQ ID NO: 1. The optimization principles include: a) select the most frequently used or higher codons according to the frequency table of Hansenula’s genetic code; b) avoid negative regulatory elements that may potentially affect gene transcription or protein translation, such as PolyAT region, PolyGC region, Silencer region and internal splice sites, etc.; c) Comprehensive analysis of mRNA secondary structure including 5' UTR, HPV45L1 coding region and 3' UTR to avoid the formation of complex RNA secondary structure, Reduce the free energy of the mRNA secondary structure; d) Use the 5'UTR region in the upstream of the coding region as much as possible that is completely consistent with the...

Embodiment 3

[0055] Example 3: Generation of Expression Constructs Carrying HPV45L1 Nucleotide Sequences

[0056] 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 the application number of 201210021524.X.

[0057] (1) PCR amplification of MOX promoter and MOX terminator

[0058] Using the mixed genomic DNA of Hansenula yeast strain ATCC26012 and ATCC34438 as a template, a MOX promoter with a size of 1518 bp was obtained by amplification with the following primer pairs, and a NotI restriction site was introduced upstream;

[0059] MOX promoter upstream primer: 5'-AAGGAAAAAAGCGGCCGCAACGATCTCCTCGAGCTGCTCGC-3' (SEQ ID NO:3)

[0060] Primer downstream of MOX promoter: 5'-TTTGTTTTGTACTTTAGATTGATGTC-3' (SEQ ID NO:4)

[0061] Using the mixed genomic DNA of Hansenula strains ATCC26012 and ATCC34438 as the template, the following primer pairs were used to amplify the MOX termina...

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Abstract

The invention relates to a method for producing HPV45 L1 protein by using a Hansenula polymorpha expression system. Specifically, the invention discloses a method for producing a recombinant Hansenula polymorpha cell expressing the HPV45 L1 protein and the recombinant Hansenula polymorpha cell produced by using the method. The invention further discloses a method for producing the HPV45 L1 protein by using the recombinant Hansenula polymorpha cell and application of the produced HPV45 L1 protein in preparation of prophylactic vaccines.

Description

Field of Invention [0001] The invention belongs to the technical field of medical bioengineering, and relates to a method for producing HPV45 L1 protein, in particular to a method for producing HPV45 L1 protein by using a Hansenula yeast expression system. Background technique [0002] Human papillomavirus (HPV) is a non-enveloped, closed-loop, double-stranded DNA virus belonging to the subfamily of Polyomaviruses of the family Papitoviridae. 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 kinds. HPV types are associated with reproductive tract infections, of which more than 20 are associated with tumors. According to the different benign and malignant lesions induced by HPV, HPV can be roughly divided into two categories: 1) High-r...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12P21/02A61K39/12A61P31/20C12R1/78
Inventor 刘娟陈丹程海王贻杰于跃李鼎锋刘勇
Owner BEIJING ABZYMO BIOSCIENCES CO LTD
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