Vaccine for preventing porcine epizootic diarrhea disease
A porcine epidemic diarrhea and vaccine technology, applied in the field of genetic engineering, can solve the problems of long antigen gene, difficult to guarantee safety, poor strain stability, etc., and achieve the effect of good stability, good protection and good safety
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Embodiment 1
[0051] Embodiment 1 Construction of recombinant bacteria of the present invention
[0052] 1 PEDV S 499-650 gene cloning
[0053] 1.1 Primer design
[0054] Referring to the S gene cDNA sequence of the PEDV SC-L strain published by GenBank (sequence number: KC886306), primers were designed using Primer 5.0 as follows (Table 1), wherein P1 / P2 were used to amplify S 499-650 Gene. Primers were synthesized by Dalian Bao Biological Engineering Co., Ltd.
[0055] Table 1 S 499-65 Gene amplification primer sequence and restriction site marking
[0056]
[0057] 1.2 PEDV S 499-650 gene amplification
[0058] 1.2.1 Extraction of PEDV RNA
[0059] Use the purified PEDV SC-L strain virus solution frozen in the laboratory as the material, and extract the viral RNA according to the instructions of the total RNA extraction kit. The operation method is as follows:
[0060] (1) Take 250 μL of virus liquid into a 2 mL EP tube, add 750 μL of RZ lysate, and beat several times with a ...
experiment example 1
[0175] Experimental example 1 Characteristic identification of recombinant attenuated Salmonella of the present invention
[0176] 1. Experimental method
[0177] Get the recombinant Salmonella SL7207 (pVAX-S) prepared in Example 1 499-650 ), according to the following method for identification:
[0178] 1 Biological characteristics of recombinant attenuated Salmonella
[0179] 1.1 Determination of growth curve of recombinant attenuated Salmonella
[0180] Pick SL7207 (pVAX-S 499-650 ) single colony, inoculated in 5mL LB liquid medium containing 100μg / mL Kan, until the bacterial solution OD 600 When the value is 0.6, inoculate 1 mL into 100 mL LB liquid medium containing 100 μg / mL Kan, culture at 37 °C, 100 r / min, for 24 h, absorb a small amount of bacterial liquid every 2 h, serially dilute and spread on three On the LB solid plate containing 100 μg / mL Kan, after culturing at 37°C for 18 hours, the colonies were counted, and the growth curves of different strains of reco...
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