Anti-Mycoplasma bovis and Pasteurella mixed protein spray
A technology for Pasteurella and Mycoplasma bovis, applied in the direction of antibody, antibacterial drug, aerosol delivery, etc., which can solve the problems of drug resistance, long diagnosis time, and inability to use drugs early
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Embodiment 1
[0014] Example 1 Md -UF1, Md -AP2 clone
[0015] 1. Use the kit (SV Total RNA Isolation) produced by Promega to extract three-day-old larvae and use the 2- and 3-day-old house flies ( Musca domestica L. ) Total RNA of larvae (species blow fly, fly eggs purchased from Liaoyuan Ecological Breeding Factory in Jilin Province, incubated by the Pharmacology and Toxicology Laboratory of Jilin Agricultural University), using a kit produced by TaKaRa Oligotex TM -dT30 mRNA Purification Kit (From Total RNA) separation and purification of mRNA;
[0016] 2. Reverse transcription synthesis of the first strand of cDNA
[0017] (1) Take two EP tubes and add the following reagents respectively, see Table 1:
[0018] Table 1 Reaction system
[0019]
[0020] (2) Mix by pipetting, centrifuge briefly, warm bath at 72°C for 3 minutes in the PCR machine, warm bath at 42°C for 2 minutes, then centrifuge briefly, add the following reagents to see
[0021]
[0022] (3) Mix well, centrifuge briefly, in a ...
Embodiment 2
[0032] Example 2 Anti-Mycoplasma Bovis, Bovine Capsular Serum Type A Pasteurella multocida Protein Md -UF1, Md -AP2 expression and purification
[0033] 1) The plasmid Md -UF1-pMD18-T, Md -AP2-pMD18-T, pET-32a(+), after EcoR I. Xho I double digestion, will Md -UF1, Md -Insert the AP2 gene into pET-32a(+) to construct a recombinant expression plasmid Md -UF1-pET-32a, Md -AP2-pET-32a;
[0034] 2) Transform into E. coli BL21 ( DE3 ), induced expression by IPTG;
[0035] 3) Purify the fusion protein using nickel ion metal chelating affinity chromatography medium (Ni-NTA Agarose).
[0036] The target protein was eluted step by step with the eluent containing 500mM imidazole to separate the protein from the column. The SDS-PAGE analysis result can be seen image 3 , Figure 4 .
[0037] The concentration of protein purified by Ni-NTA Agarose column was measured by NANO DROP 2000 with a trace ultraviolet spectrophotometer as follows: Md -UF1-pET-32a and Md -AP2-pET-32a protein con...
Embodiment 3
[0038] Example 3 Md -UF1, Md -Preparation of AP2 mixed protein gel spray
[0039] In this study, an ion-sensitive instant gel matrix was selected according to the environmental characteristics of the nasal cavity, that is, desacetyl gellan gum, as the coagulation matrix, and the prescribed amount of antimicrobial peptides and solvents were added and stirred together with the excipients to make a suspension. The agent contains 0.8mg of mixed protein. The specific composition is shown in Table 3. The mixing steps are as follows:
[0040]
[0041] Weigh the prescription amount of acetyl gellan gum into the container according to Table 3, add an appropriate amount of deionized water, heat it in a water bath at 100°C and keep stirring until it is evenly dispersed, and then place it in a 4°C refrigerator to completely cool for later use, add the prescription Measure the antibacterial protein, mannitol, and chloretidine and stir to dissolve them. Finally, add deionized water to the tot...
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