Selenium-enriched yeast gene engineering bacteria and surface display system thereof and construction method of surface display system

A surface display system and genetically engineered bacteria technology, applied in the field of molecular biology, can solve problems such as timeliness, no suitable medicine or vaccine available for epidemic diseases, quality problems, etc., and achieve the effect of broadening the application range and enhancing the body's immunity

Pending Publication Date: 2021-08-31
CHONGQING ACAD OF ANIMAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, some vaccine products have many disadvantages such as quality problems and timeliness problems, and there are even no suitable drugs or vaccines available for some diseases, especially diseases caused by new strains and mutant strains of pathogenic microorganisms that are constantly emerging. Prevention and control of goat diseases The situation is grim, therefore, it is particularly important to improve the immunity and disease resistance of goats

Method used

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  • Selenium-enriched yeast gene engineering bacteria and surface display system thereof and construction method of surface display system
  • Selenium-enriched yeast gene engineering bacteria and surface display system thereof and construction method of surface display system
  • Selenium-enriched yeast gene engineering bacteria and surface display system thereof and construction method of surface display system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Design and synthesis of CapIFN-γ target gene

[0042] (1) Design of the target gene

[0043] Since the pYD1 plasmid still maintains its inherent V5 epitope and Histag expression tags after Kpn I / EcoR I double digestion, in order to better investigate the expression site and expression level of the target protein in the follow-up, the CapIFN-γ mature peptide gene sequence A His tag sequence is added to the downstream primer, and the stop codon TAA is designed to prevent the expression of V5 epitope and His tag, and the target protein can be screened and identified based on the His tag. Therefore, the target gene sequence is mainly composed of KpnI, CapIFN-γ, His tag, and EcoR I gene sequences. The full length of the gene is 469bp, and the target gene is optimized and designed according to the yeast codon preference.

[0044] CapIFN-γ mature peptide gene sequence information:

[0045] The size is 440bp, and the KpnI restriction endonuclease gene (CGGGTACC) is ...

Embodiment 2

[0054] Example 2 Construction of pYD1-CapIFN-γ recombinant shuttle plasmid

[0055] (1) Extraction and digestion of plasmid

[0056] refer to figure 1 , Extract the pYD1 shuttle plasmid and the pUC57-CapIFN-γ recombinant cloning plasmid according to the instructions of the plasmid mini-extraction kit, and perform double digestion with KpnI and EcoRI restriction endonucleases respectively. The 20 μL enzyme digestion system is: KpnI / EcoRI 1 μL each , Buffer 2 μL, plasmid 12 μL, ddH2O 4 μL, 37 ° C water bath for 1.5 h, after the end, perform electrophoresis analysis on 1.0% agarose gel, and refer to the instructions of the DNA agarose gel recovery kit to digest the pYD1 plasmid and the target fragment After recovery and dephosphorylation, store at -20°C for future use.

[0057] (2) Connection and conversion

[0058] Use T4 DNA ligase to connect the target fragment and pYD1 plasmid. The 10 μL ligation system is: T4 DNA ligase 1 μL, Buffer 5 μL, target fragment 3 μL, pYD11 μL, d...

Embodiment 3

[0069] Example 3 Selenium-enriched transformation of Saccharomyces cerevisiae and preparation of competent cells

[0070] (1) Expanded culture of Saccharomyces cerevisiae and drawing of growth curve

[0071] Pick Saccharomyces cerevisiae EBY100 and inoculate it in YPD solid medium, and place it in a constant temperature incubator at 30°C for cultivation. After a single colony grows, pick a single colony and inoculate it in 10mL of YPD liquid culture, and place it for constant temperature cultivation at 30°C In the box, shake culture at 250r / min, culture for 24h, then inoculate 10% of the inoculum into 200mL YPD fresh medium, sample 1mL every 12h, measure the content of yeast, culture continuously for 72h, and plot according to the bacterial content at each time point its growth curve. It was found that the cell content of Saccharomyces cerevisiae EBY100 reached the peak when it was cultured for 36 hours, and the cell content reached 210×106 CFU·mL-1.

[0072] (2) Selenium-en...

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Abstract

The invention belongs to the field of molecular biology, and particularly relates to selenium-enriched yeast gene engineering bacteria and a surface display system thereof and a construction method of the surface display system. Yeast gene engineering bacteria are cultured by using a culture medium, and then sodium selenite is added for continuous culture to obtain the selenium-enriched yeast gene engineering bacteria. Goat gamma-interferon is inserted into a His tag, and pYD1-CapIFN-gamma is constructed by taking a pYD1 shuttle plasmid as a skeleton; and then the pYD1-CapIFN-gamma is introduced into the selenium-enriched yeast gene engineering bacteria to obtain a goat gamma-interferon selenium-enriched yeast display system. The system carries out selenium-enriched transformation on the yeast in the system, so that the application range of a goat gamma-interferon yeast expression system is widened; the yeast surface display of the goat gamma-interferon is implemented, which is more beneficial for exerting the antiviral activity of the goat gamma-interferon; and a foundation is laid for research and development of a safer, more effective and cheaper novel yeast feed additive capable of resisting to viruses and enhancing the immunity of organisms.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to a selenium-enriched yeast genetically engineered bacterium, a surface display system and a construction method thereof. Background technique [0002] Interferon (interferon, IFN) is a non-specific broad-spectrum anti-virus induced protein secreted by cells, mainly glycoproteins, and its main biological functions include: broad-spectrum anti-virus, immune regulation, immune enhancement and anti-tumor, etc. . Among them, veterinary interferon has become a research hotspot due to its unique antiviral activity and immune regulation effect. There are relatively many studies on human, pig, cow, poultry and dog interferon. Interferon produced by genetic engineering has been widely used in Prevention, control and treatment of animal diseases such as poultry, pigs and dogs. Natural interferon is only produced under specific induction conditions, and its content is low, so it ...

Claims

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Application Information

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IPC IPC(8): C12N1/36C12N1/19C12N15/23C12N15/81C07K14/57C12R1/84C12R1/865C12R1/645
CPCC12N1/36C12N15/81C12N15/815C07K14/57C12N2800/102
Inventor 许国洋杨睿高广亮杨柳郑华余远迪牟豪闫志强张素辉
Owner CHONGQING ACAD OF ANIMAL SCI
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