Yersinia pestis ghost, preparation method thereof, and application of Yersinia pestis ghost serving as vaccine
A technology of Yersinia and plague, applied in the fields of molecular biology and immunology, can solve the problems of high side reaction rate, poor production safety, and single ingredient, and achieve simple and easy preparation process, reduced possibility, strong The effect of immune memory
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Embodiment 1
[0037] Preparation and identification of embodiment 1 plague EV slough
[0038] 1.1 Electrotransformation of Yersinia pestis with plasmid pMBE
[0039] The production method of EV competent cells is as follows: Inoculate a single plague EV colony into 5ml LB liquid medium, culture overnight at 37°C, then expand and transfer to 100ml LB medium, culture at 37°C until mid-logarithmic growth, and collect the bacteria by centrifugation Body, wash twice with PBS (PH7.4) and electrotransfer medium (0.5M sorbitol, 0.5M mannitol, 10% glucose, distilled water to volume), and finally add 1ml pre-cooled electroporation medium, 100ul aliquot , stored at -70°C.
[0040] The operation method of electroporation is as follows: After mixing the correctly identified plasmid pMBE with 100 μl of competent cells, inject it into a pre-cooled 0.1 cm electric shock cup (BIO-RAD), and set on the electric shock instrument: voltage 1500v, capacitance 25uF, resistance 200Ω , for electrical conversion. ...
experiment example 2
[0053] Experimental Example 2 Analysis of Immune Effect of Plague EV Sludge
[0054] BALB / c mice were immunized with plague EV slough by subcutaneous injection, and the immune response and immune protection induced by the slough were analyzed.
[0055] 2.1 Experimental animals and grouping
[0056] Healthy BALB / c female mice, 6-8 weeks old, weighing about 18 g, were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences. Randomly divided into 3 groups, 5-6 in each group. In the experimental group (EV slough group), the freeze-dried slough was directly dissolved in PBS without adding any adjuvant, and each mouse was subcutaneously inoculated with 10 7 CFU / 100μL, a total of two immunizations (boosting immunization on the 21st day); at the same time, a positive control group (EV live vaccine group, Lanzhou Institute of Biological Products produced human plague EV live vaccine) was immunized once, and each mouse was vaccinated dose of 10 7 CF...
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