Mycoplasma bovis Mbov_0570 gene mutant strain and application thereof
A technology of Mycoplasma bovis and gene is applied in the field of Mycoplasma bovis Mbov_0570 gene mutant strain to prepare Mycoplasma bovis vaccines, which can solve problems such as unclear pathogenic mechanism, hindering effective prevention, control and treatment of Mycoplasma bovis, and hindering the development of new drugs and new vaccines.
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Embodiment 1
[0010] Embodiment 1: Construction of Mycoplasma bovis insertion mutant library
[0011] The applicant isolated a strain of Mycoplasma bovis HB0801 from the diseased lung tissue of sick cattle in June 2008, and named it Mycoplasma bovis HB0801 (Mycoplasma bovis HB0801). This strain has been disclosed in the patent document of CN 102220263A.
[0012] The pMT85 plasmid was donated by Dr. Eric Baranowski of the French Academy of Agricultural Sciences in France. It contains a mini-type Tn4001 transposon (mini-Tn4001), and the gentamicin resistance marker encoded by the aacA-aphD gene has been introduced into the transposon. Located between two inverted repeats (IRs) at both ends of the transposable fragment, the transposase gene (tnpA) is located outside the repeats and prevents transposition from occurring again (Baranowski et al 2010).
[0013] Using M.bovis HB0801 as the parent strain to construct a mutant library, the basic procedure is as follows: collect M.bovis cultured to t...
Embodiment 2
[0014] Embodiment 2: Screening and identification of Mycoplasma bovis T9.171 mutant
[0015] Using the bacterial genome extraction kit (purchased from Bao Bioengineering Dalian Co., Ltd.), the whole genome of the Mycoplasma bovis T9.171 mutant was extracted, and the junction of the Tn4001 transposon and the Mycoplasma bovis genome was sequenced. The genome sequence was compared, and the results showed that the T9.171 mutant gene was Mbov_0570, and the transposon insertion site was located after the 672416 site of the genome and after the 409 site of the Mbov_0570 gene, with a size of 3438bp ( figure 1 ).
Embodiment 3
[0016] Example 3: T9.171 mutant stimulates BoMac cells to reduce the ability to express cytokines
[0017] Culture and counting of Mycoplasma bovis: Take Mycoplasma bovis HB0801, Mycoplasma bovis MbovHB0801-150.2, and T9.171 to inoculate PPLO liquid medium at a ratio of 1:1000, and place it at 37°C, 5% CO 2 After culturing in the incubator for 36 hours to reach the logarithmic phase, count the CFUs. The method is as follows: Dilute the cultured bacterial solution by 10 times, take 10 μL of the appropriate dilution of the bacterial solution and spread it on the PPLO solid medium, at 37 ° C, Inverted culture, 5% CO 2 After culturing in the incubator for 3-7 days, count the colonies under a stereo microscope. The formula for calculating the number of colonies is: CFU / mL=number of colonies×dilution×100.
[0018] Place the BoMac in a 2×10 6 The cell density of each well was inoculated into a 6-well cell plate, cultured at 37°C, 5% CO2 until adherent, and added Mycoplasma bovis HB...
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