Preparation method of mycoplasma bovis immunomagnetic beads

A technology of Mycoplasma bovis and magnetic beads, which is applied in the field of preparation of Mycoplasma bovis immune magnetic beads, can solve the problems of long detection time and low detection rate of Mycoplasma bovis

Inactive Publication Date: 2020-07-31
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the invention is to solve the problem of long detection time and low detection rate of Mycoplasma bovis, and provide a preparation method of Mycoplasma bovis immune magnetic beads

Method used

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  • Preparation method of mycoplasma bovis immunomagnetic beads
  • Preparation method of mycoplasma bovis immunomagnetic beads
  • Preparation method of mycoplasma bovis immunomagnetic beads

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Cloning of Mycoplasma Bovis Gene

[0038] The designed primer sequence was sent to Shenggong Bioengineering (Shanghai) Co., Ltd. for synthesis, and two restriction sites, BamHI and XhoⅠ, were added. The primer sequence is shown in Table 1; the P48 gene was analyzed using the Mycoplasma bovis genome Cloning; reaction conditions are shown in Table 2; 50μL PCR reaction system:

[0039] Template DNA 4.0μL

[0040] Upstream primer (20pmol / μL) 1.0μL

[0041] Downstream primer (20pmol / μL) 1.0μL

[0042] 10×Ex Taq Buffer (Mg2+Plus) 5.0μL

[0043] dNTP Mixture (2.5mM each) 4.0μL

[0044] Ex Taq (5U / μL) 0.6μL

[0045] ddH20 34.4μL.

[0046]

[0047]

[0048] The PCR products were subjected to 1% and 0.5% agarose gel electrophoresis, and the gel imaging analyzer was used to detect the PCR amplification results; the PCR products were recovered with the gel recovery kit according to the instructions of the gel recovery kit, and then combined with pmd-18T The vector was ligated and lig...

Embodiment 2

[0050] Example 2 Construction of expression vector of Mycoplasma bovis protein

[0051] Extract the plasmids from the above-sequenced strains and strains containing pET32a(+) empty plasmids and double-enzyme digestion with BamHI and XhoI restriction enzymes, and perform agarose gel electrophoresis on the digested products to recover the target gene strip of Mycoplasma bovis P48 Band and pET32a(+) empty plasmid band; use T4 ligase to ligate the P48 gene and pET32a(+) vector at 16℃; the ligation product was transferred to E. coli DH5α competent cells. Cultivate for 12-14h on selective plates containing ampicillin resistance, pick a single colony, extract plasmids and perform double enzyme digestion verification, send the above correct strains to Shanghai Shenggong Bioengineering (Shanghai) Co., Ltd. for sequencing Identification.

[0052] The constructed recombinant expression plasmid pET32a-P48 was subjected to double enzyme digestion (BamHI and XhoⅠ) for verification; the digested...

Embodiment 3

[0053] Example 3 Expression and purification of recombinant P48 protein

[0054] The correct strain was extracted from the plasmid and transformed into E. coli BL21 (DE3) competent cells, and cultured on selective plates containing ampicillin resistance for 12-14h. Select the recombined correct strains in a liquid medium containing ampicillin-resistant LB, and cultivate overnight at 37°C and 220r / min. Inoculate the bacterial solution that has been cultured overnight in 300 mL of medium at a ratio of 1:100, cultivate until the OD value of the bacterial solution reaches 0.4-0.6, add ITPG with a final concentration of 1 mmol / L, and induce overnight at 16°C. Centrifuge for 10 minutes at 8000r / min and 4°C. After the cells were washed several times with sterilized PBS, the cells were resuspended in 20 ml of PBS and placed on ice for ultrasonic disruption. Afterwards, the crushed product was centrifuged at 12000 r / min and 4°C. Take the supernatant and precipitate after centrifugation...

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Abstract

The invention discloses a mycoplasma bovis P48 gene. The base sequence of the mycoplasma bovis P48 gene is shown as SEQ ID NO. 1 in a sequence table; a mycoplasma bovis P48 antigen protein is a protein expressed by the mycoplasma bovis P48 gene; an anti-mycoplasma bovis polyclonal antibody is a polyclonal antibody prepared by taking the mycoplasma bovis P48 antigen protein as an antigen; and a preparation method of an anti-mycoplasma bovis immunomagnetic bead is disclosed. Results show that 1) the coupling efficiency of NHS magnetic microspheres is highest when a coupling buffer solution is 2-morpholineethanesulfonic acid (MES) and the antibody concentration is 200 [mu] g/mL; and 2) the prepared immunomagnetic beads can be enriched into mycoplasma bovis within 15 minutes, the lowest enrichment concentrations are that the bacterium concentration is 10<-3>CCU/mL, and the immunomagnetic beads have good specificity and sensitivity.

Description

Technical field [0001] The invention belongs to the technical field of immune magnetic beads preparation, and specifically relates to a preparation method of Mycoplasma bovis immune magnetic beads. Background technique [0002] Mycoplasma bovis ( Mycoplasma bovis, M.bovis ) Belongs to the kingdom of Prokaryotes, Periphyta, Choroids, Mycoplasma, Mycoplasmaceae, and Mycoplasmaceae, which is one of the most pathogenic species in the Mycoplasma genus. my country After Li Jishen first reported the isolation of Mycoplasma bovis from cow's milk in 1983, in 2008, Xin Jiuqing and others isolated the pathogenic microorganism from lungs with bovine pneumonia for the first time. Mycoplasma bovis can cause diseases such as pneumonia, mastitis, arthritis, reproductive organs and keratitis in cattle. The diseases it causes mostly occur in fattening shelf cattle that have been transported over long distances, causing serious economic losses to the animal husbandry industry in my country. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/30C07K16/12G01N33/543G01N33/569C12R1/35
CPCC07K14/30C07K16/1253G01N33/54326G01N33/56933G01N2333/30
Inventor 孔令聪马红霞翟志超王雪
Owner JILIN AGRICULTURAL UNIV
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