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Swine fever virus subunit vaccine and preparation method and purpose thereof

A swine fever virus and swine fever subunit technology, which is applied in the field of swine fever virus recombinant subunit vaccine and its preparation, can solve problems such as the inability to completely prevent vertical transmission and the like

Active Publication Date: 2018-08-14
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

E2 subunit vaccines can reduce vertical transmission, but cannot completely prevent vertical transmission. After E2 subunit vaccination, there are differences in infection and inoculation. This difference is conducive to rapid screening of infected pigs, helping to control and eliminate CSFV

Method used

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  • Swine fever virus subunit vaccine and preparation method and purpose thereof
  • Swine fever virus subunit vaccine and preparation method and purpose thereof
  • Swine fever virus subunit vaccine and preparation method and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the synthesis and the amplification of the dna sequence of coding swine fever virus mE2 protein

[0053] Comparing the popular strains of classical swine fever virus at home and abroad, the CSFV subtype 2.1b strain (Classical Swine Fever Virus strain Hun23 / 2013, GenBank: KP233071.1) popular in my country was selected. E2 The gene sequence is used as the gene source of the recombinantly expressed classical swine fever virus E2 protein sequence of the present invention.

[0054] According to literature reports, the main antigenic epitope of CSFV is located in the 1-176th position of the E2 protein, so the present invention selects the 1-678th nucleotide sequence of the E2 gene of the CSFV 2.1b strain for recombination express and name mE2 Gene. Referring to the codon preference of Kluyveromyces marx, without changing the amino acid sequence, the mE2 The nucleotide sequence of the gene is codon-optimized, and after optimization mE2 The nucleotide sequence...

Embodiment 2

[0056] Example 2, Construction of Kluyveromyces marx recombinant expression vector for mE2 protein of classical swine fever virus

[0057] restriction enzyme Speech I and Sac II Carry out double enzyme digestion on the Kluyveromyces marx expression vector pUKDN125, and after the digested product undergoes 1% agarose gel electrophoresis, the vector fragment of about 7.2kb is recovered with the SanPrep column DNA gel recovery kit. Using Gibson Assembly traceless connection system (NEB company, item number E2611S / L), the mE2 The gene segment is connected with the carrier segment to obtain the recombinant expression vector pUKDN125-mE2 of the CSFV mE2 protein Kluyveromyces marx. The recombinant vector includes yeast autonomous replication sequence, inulinase promoter, classical swine fever virus mE2 Genes, inulinase terminators, selection marker genes URA 3 promoters and their promoters.

Embodiment 3

[0058] Example 3, Construction of Kluyveromyces marx recombinant strain Fim-1-mE2

[0059] The yeast expression host strain used in the present invention is derived from Kluyveromyces marxense strain Fim-1 (CGMCC No. 10621 of the China General Microorganism Culture Collection Management Center), which is knocked out by homologous recombination. URA3 Gene, and use the YPD containing 5 fluoroorotic acid (1.5g / L) to screen to obtain the expression host bacterium of uracil deficiency, named as Kluyveromyces marxis Fim-1 ura3∆ . Transform pUKDN125-mE2 into Fim-1 using lithium acetate conversion method (World Journal of Microbiology & Biotechnology 16: 653-654, 2000) ura3∆ middle. Spread the transformed product on an SD plate (0.67% amino acid-free yeast nitrogen source, 2% glucose, 2% agar), place the plate in a constant temperature incubator at 30°C and culture it for 2-4 days until colonies are formed.

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Abstract

The invention belongs to the technical field of biological medicine, and concretely relates to a swine fever virus subunit vaccine and a preparation method and a purpose thereof. The invention provides a kluyveromyces marxianus yeast recombination strain used for preparing the swine fever virus subunit vaccine. The recombination strain is constructed by the following steps: swine fever virus envelope protein E2 is intercepted, through codon optimization, a coding sequence of the swine fever virus mE2 protein is obtained, and then is cloned to a kluyveromyces marxianus yeast expression vector,and the kluyveromyces marxianus yeast host strain is transformed. The invention also provides the method for preparing the swine fever virus subunit vaccine, which comprises the following steps: the kluyveromyces marxianus yeast host strain is subjected to recombination expression by using mE2, steps of culture, centrifugation, cell disruption, and separating purifying are carried out to obtain the swine fever virus mE2 protein antigen, and the purified antigen and an adjuvant are subjected to complex formulation to prepare the swine fever virus subunit vaccine. The injection immunotherapy ofswine fever virus mE2 protein recombination subunit vaccine can obtain a protective IgG antibody, and the subunit vaccine can reduce and prevent the swine fever virus infection-related disease.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to the recombinant preparation of the envelope protein mE2 of classical swine fever virus by Kluyveromyces marxe; it also relates to a recombinant subunit vaccine of classical swine fever virus and its preparation method and application. Background technique [0002] Swine Fever (SF), also known as swine cholera or rotten intestinal fever, is also called Classical Swine Fever (CSF) in Europe in order to distinguish it from African swine fever. Classical swine fever is a highly contagious disease of pigs caused by classical swine fever virus (CSFV). It is mainly characterized by acute onset, high fever, degeneration of small blood vessels, causing hemorrhage, infarction, necrosis and high mortality. CSFV belongs to the family Flaviviridae, a member of the genus Pestivirus. This virus genus also includes bovine viral diarrhea virus (BVDV) and sheep border disease viru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C07K14/18A61K39/187A61P31/14C12R1/645
CPCA61K39/12A61K2039/552A61P31/14C07K14/005C12N15/815C12N2770/24322C12N2770/24334
Inventor 吕红朱培霞周峻岗余垚
Owner FUDAN UNIV
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