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Recombinant hansenula polymorpha capable of expressing Zika virus E protein under assistance of molecular chaperone and construction method of recombinant hansenula polymorpha

A technology of Hansenula polymorpha and Zika virus, applied in the field of genetic engineering, can solve the problems of non-expression and low efficiency of exogenous expression of ZKE protein, and achieve the effects of low cost, easy high-density fermentation, and simple purification steps

Inactive Publication Date: 2018-06-08
NINGBO MUNICIPAL CENT FOR DISEASE CONTROL & PREVENTION +1
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Problems solved by technology

The main difficulty may be that the secretory expression of the protein depends on an extra sequence at the N-terminal of the protein and whether the protein can be folded correctly. However, affected by the structure and folding of the ZKE protein, the exogenous expression efficiency of the ZKE protein in the host cell is low or even not expressed

Method used

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  • Recombinant hansenula polymorpha capable of expressing Zika virus E protein under assistance of molecular chaperone and construction method of recombinant hansenula polymorpha
  • Recombinant hansenula polymorpha capable of expressing Zika virus E protein under assistance of molecular chaperone and construction method of recombinant hansenula polymorpha

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Embodiment Construction

[0026] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

[0027] In the present invention, the Zika virus structural protein E gene fragment without signal peptide and the calnexin (CNE1) gene (HpCne1) fragment derived from Hansenula polymorpha (H.polymorpha) were respectively cloned into the plasmid pHMOXG- In alpha-A (or called pHMOXG-α-A), a recombinant co-expression vector pHMOXG-α-ZKE-HpCne1 was constructed, which was transfected into Hansenula polymorpha DL-1 (host cell), and cultured and induced, The high-throughput secretion and expression of recombinant Zika virus structural protein E by host cells has been realized.

[0028] (1) Construction of vector pHMOXG-α-ZKE-HpCne1 and transformation of H.polymorpha DL-1

[0029] Restriction endonucleases were purchased from Takara, and the primer sequences used are shown in Table 1:

[0030] Table 1. Primer sequences used in the experiment

[0031...

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Abstract

The invention provides recombinant hansenula polymorpha capable of expressing Zika virus E protein under the assistance of a molecular chaperone and a construction method of the recombinant hansenulapolymorpha. The construction method comprises the following steps: firstly, constructing a coexpression vector of recombinant Zika virus structural protein E and molecular chaperone hansenula polymorpha calcium connexin, wherein the sequence of the recombinant Zika virus structural protein E is shown as SEQ.ID.NO.1 and the gene fragment of the hansenula polymorpha calcium connexin is shown as SEQ.ID.NO.2; and secondly, transforming the linearized coexpression vector into a hansenula polymorpha cell through electroporation transformation, performing induction culture, purifying a recombinant hansenula polymorpha fermentation product, and then detecting that the recombinant protein content can reach 12.6 mg / L.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the establishment of a Hansenula polymorpha expression system for Zika virus glycoprotein gene. Background technique [0002] Zika virus (Zika virus) belongs to the family Flaviviridae (Flaviviridae), which is similar to dengue virus, yellow fever virus, Japanese encephalitis virus, and West Nile virus. Zika virus is a single-stranded positive strand RNA Viruses, the infection rate has increased in recent years. The prevalence of Zika virus not only seriously threatens the lives of the people, but also causes serious psychological impact and heavy economic burden. [0003] The Zika virus genome is about 10.8kb long and contains a single open reading frame. The viral protein consists of a single polyprotein precursor, which is digested by host protease and viral protein. The viral protein includes three structural proteins (C, prM / M, E) and seven nonstructural proteins (NS1, N...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12N15/40C07K14/18C12R1/78
CPCC07K14/005C12N15/815C12N2770/24122
Inventor 李永东钱卫东刘昱慧焦素黎倪红霞
Owner NINGBO MUNICIPAL CENT FOR DISEASE CONTROL & PREVENTION
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