Application of rainbow trout IFN-gamma2 (Interferon-gamma2) in preparing anti-IHN (Infectious Haematopoietic Necrosis) virus product
A virus and product technology, applied in the application field of rainbow trout IFN-γ2 in the preparation of anti-IHN virus products, can solve the problems of recessive infection, high mortality of fry or juvenile fish, etc.
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Embodiment 1
[0026] Example 1. Obtaining of rainbow trout IFN-γ2 gene and its recombinant vector for expression
[0027] 1. Cloning of the target gene
[0028] The rainbow trout (Oncorhynchus mykiss, kept and bred in the laboratory, a conventional rainbow trout species) was killed, and the head kidney and spleen were quickly aseptically stripped on ice, and an appropriate amount of MEM medium containing 10% FBS was added. Filtered through a 70 μm nylon mesh (product of Fisher Scientific), stimulated by 5 μg / mL phytohemagglutinin, and extracted total RNA using the RNA extraction kit of Promega Company, according to the instructions of M-MLV reverse transcriptase, with primer Oligo( dT)18 was reverse transcribed to obtain the first strand of cDNA.
[0029] Referring to the published rainbow trout IFN-γ2 sequence (Genbank: FM864345.1), design upstream and downstream primers for amplifying rtIFN-γ2, upstream primer: 5'-ATC GGATCC GCTCAGTACACATCAATTAAC-3' (underlined BamHI restriction site),...
Embodiment 2
[0040] Embodiment 2, application of rainbow trout IFN-γ2 gene
[0041] 1. Protein expression and expression pattern analysis
[0042]The recombinant strain DH5α / pET32a-rtIFN-γ2 was cultured overnight in 5 mL LB medium containing 100 μg / mL AMP at 37° C. with shaking at 220 rpm. The next day, the overnight culture was inoculated in 100 mL LB medium at a ratio of 1:100, cultured with shaking at 220 rpm until the OD value reached 0.6-0.8, and the expression was induced by adding 1 mM IPTG. After 4 hours, the cells were collected by centrifugation at 4000 rpm at 4 °C.
[0043] The bacteria were resuspended in PBS with 1 / 10 of the initial medium volume, and divided into supernatant and precipitate after ultrasonic lysis and crushing. SDS-PAGE was used to analyze the existing form of the target protein.
[0044] The result is as figure 2 As shown, M is the protein molecular mass standard; 1 is before the induction of the expression strain; 2 is after the induction of the expressio...
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