Pichia pastoris for expressing rotavirus expression particles as well as preparation method and application of pichia pastoris

A rotavirus and virus-like particle technology, applied in the field of genetic engineering, can solve problems such as side effects, intussusception, and weak cross-protection of vaccines

Active Publication Date: 2012-05-23
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently marketed rotavirus vaccines are mainly live vaccines of human-animal reassortant strains and animal attenuated strains. The application of such vaccines can lead to serious side effects such as intussusception
Infection caused by different serotypes of rotavirus, the cross-protection of the vaccine may be weak, and the severe side effects of the current vaccine may cause a strong need for a new rotavirus vaccine to meet the needs of treatment and prevention

Method used

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  • Pichia pastoris for expressing rotavirus expression particles as well as preparation method and application of pichia pastoris
  • Pichia pastoris for expressing rotavirus expression particles as well as preparation method and application of pichia pastoris
  • Pichia pastoris for expressing rotavirus expression particles as well as preparation method and application of pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0048] 2. Antibody preparation:

[0049] After obtaining intact serotype-specific VLPs, they are recovered by purification and used to vaccinate individuals to obtain protective immunity. This method of immunization and studies to verify immunity can be found in the following literature. [1] Offit PA, Dudzik KI. Noninfectious rotavirus (strain RRV) induces an immune response in mice which protects against rotavirus infection against rotavirus challgenge.) Journal of Clinical Microbiology, 1989, 27(5): 885-888; [2] McNeal MM, Sheridan JF, Ward RL. The protective effect of intraperitoneal immunization against mouse rotavirus infection (Active protection against Rotavirus infection of mice following intraperitoneal immunization.) Journal of Virology, 1992, 191(1): 150-157; [3] Ciarlet M, Crawford SE, Barone C, etc. Rotavirus subunit vaccine by parenteral immunization experts Rabbit-induced protective immunity (Subunit rotavirus vaccine administered parenterally to rabbits induc...

Embodiment 1

[0063] Example 1 Optimization and synthesis of rotavirus VP protein structural gene

[0064] Through the analysis of Blast software and the comparison of nucleotide and amino acid homology, Chinese local human rotavirus strains with greater homology with international standard strains and Asian strains were selected as VP2, VP6, VP4 (P8 serotypes) ), VP7 (G1 and G3 serotypes) DNA template strands (Table 1). According to the Pichia pastoris codon usage frequency table (Table 2), the DNA sequence was synonymously substituted for all target genes, and some AT-rich regions were removed, and the GC content was adjusted to 45-50%. At the same time, synonymous mutations were carried out at the enzyme cutting sites (EcoRI, NotI, MluI, BglII, BspEI and BamHI) related to the construction of recombinant plasmids. A Kozak (ACCATG or ACCGCCATG) sequence was added to the 5' end, and a stop codon was added to the 3' end. The optimized structural genes of each VP protein were sent to Nanjin...

Embodiment 2

[0078] Embodiment 2 Transformation of pPIC3.5K expression plasmid

[0079] Design 2 primers: forward primer: 5' AGATCT TACGTAGAATTCCCTAGGGCGGCCGCGAATTAATTC-3' (SEQ ID NO: 6), reverse primer 5'GGCGAATTAATTC GGGCCC - ACGCGT - GGATCC ATCGATAAGC (SEQ ID NO: 7), the underlines are restriction sites BglII, Bsp120I, MluI and BamHI, respectively. Send to Shanghai Sangon Bioengineering Technology Service Co., Ltd. for synthesis. The pPIC3.5K plasmid was used as a template for PCR amplification with forward and reverse primers to obtain the BglⅡ-TT-Bsp120Ⅰ fragment. Using the same tail enzyme technology, the BglII-TT-Bsp120I fragment was directional cloned into the multiple cloning sites BamHI and NotI of the pPIC3.5K plasmid through the BglII and Bsp120I sites to form BamHI and MluI at the 3' end of the TT (transcription termination signal) site pPIC3.5K plasmid (to facilitate the construction of multi-copy plasmids).

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Abstract

The invention relates to pichia pastoris for expressing rotavirus expression particles as well as a preparation method and application of the pichia pastoris, and discloses a human rotavirus structural protein expression cassette, comprising the following elements: (a) an initial signal element AOX; (b) a rotavirus structural protein gene; and (c) a termination signal element TT. The invention further discloses a yeast cell transfected by using the expression cassette, a method for preparing the rotavirus viroid particles by using the yeast cell, viroid particles prepared by using the method, and application of the viroid particles for preparing vaccine compositions for preventing or treating rotavirus infection. The invention also discloses a preparation method of the yeast cell.

Description

field of invention [0001] The invention relates to the field of genetic engineering, in particular, using yeast system to express rotavirus structural protein to assemble rotavirus virus-like particles for preventing rotavirus infection. Background of the invention [0002] Rotavirus is one of the most common pathogens of severe diarrhea in infants and young children worldwide. It mainly infects small intestinal epithelial cells, causing cell damage and diarrhea. Rotavirus is prevalent in summer, autumn and winter every year. The route of infection is the fecal-oral route. The clinical manifestations are acute gastroenteritis and osmotic diarrhea. Days, severe dehydration symptoms. [0003] There are seven types of rotavirus, which are numbered A, B, C, D, E, F, and G in English letters. Humans are mainly infected by rotaviruses A, B and C, the most common of which is rotavirus A. All seven rotaviruses can cause disease in other animals. [0004] Among rotavirus A specie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/46C12N1/19C12N7/01A61K39/15A61P31/12C12R1/84
Inventor 张群竺满溢楼觉人
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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