Co-immunoprecipitation method suitable for nosema bombycis

A technology of co-immunoprecipitation and microsporidia, which is used in material testing products, measuring devices, instruments, etc., can solve problems such as non-specific bands, influence experimental results, and binding, and achieve improved specificity, high stability, and repeatability. good effect

Inactive Publication Date: 2017-03-22
CHONGQING THREE GORGES MEDICAL COLLEGE
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Problems solved by technology

[0004] However, this classic technique has deficiencies in the research on the interaction proteins of N. silkworm, mainly in three aspects: First, it is difficult to extract the total spore protein used in co-immunoprecipitation experiments. The spore wall itself is thick, and the ordinary protein extraction solution cannot penetrate deep into the spores, nor can it fully extract the protein; second, the immunoglobulin beads used for co-immunoprecipitation will naturally sink to the bottom

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  • Co-immunoprecipitation method suitable for nosema bombycis
  • Co-immunoprecipitation method suitable for nosema bombycis
  • Co-immunoprecipitation method suitable for nosema bombycis

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Embodiment 1

[0036] A method suitable for co-immunoprecipitation of microsporidium silkworm, the specific steps are as follows:

[0037] 1) Take 1×10 9个 Bombyx mori spores were mixed with 0.01mol / L PBS buffer solution with a pH of 7.2, and 0.4g of glass beads with a diameter of 0.15-0.21mm were added, shaken vigorously in a shaker at 4°C for 4h, and then placed in The supernatant obtained by centrifuging at 12000rpm for 10min is the spore cell lysate protein;

[0038]2) Take 2 μL of mouse antibody and dilute it to 500 μL with PBS buffer solution with pH 7.2, add 40 μL of Protein A+G bead suspension, and shake gently at 4°C overnight; then centrifuge at 2500 rpm for 5 min, remove the supernatant Keep the precipitate as much as possible, add 200 μL of the spore cell lysate protein prepared in step 1) to the precipitate, resuspend, place horizontally and bury it in ice at 4°C and shake slowly for 7-8 hours. During this period, replace with fresh ice cubes and shake gently. Suspend to ensure...

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Abstract

The invention discloses a co-immunoprecipitation method suitable for nosema bombycis. The three technical problems in the prior art are solved, wherein as the first problem, spore total protein for a co-immunoprecipitation experiment is difficult to extract, and as the sporoderm of the nosema bombycis is thick, common protein extraction liquid cannot enter a spore, and protein cannot be fully extracted; as the second problem, due to the gravity, immune ball beads for co-immunoprecipitation can be naturally sunk to the bottom in the reacting process, stacked ball beads, protein liquid and antibodies cannot be fully combined, and the experiment result is influenced; as the third problem, most antibodies for the co-immunoprecipitation experiment are self-prepared polyclonal antibodies, the specific effect of the antibodies is poor, and nonspecific bands usually appear; in addition, detection to target protein can also be influenced by heavy chains and light chains of the antibodies. By means of the co-immunoprecipitation method suitable for the nosema bombycis, the specificity of the nosema-bombycis co-immunoprecipitation is improved, stability is high, and repeatability is good.

Description

technical field [0001] The invention belongs to the technical field of molecular biology experiments, and in particular relates to a method suitable for co-immunoprecipitation of the microsporidium silkworm. Background technique [0002] Nosema bombycis is the causative agent of silkworm microsporidia, which belongs to obligate intracellular parasitic eukaryotes. Because it can cause great harm to sericulture through vertical transmission, it is listed as a legal quarantine object for sericulture production by sericulture countries and regions. However, how to effectively solve the quarantine and prevention of microparticle diseases has become a difficult problem in current research. With the completion of the sequencing data of the silkworm Microsporidia genome, the study of Microsporidia has entered the field of molecular level, and some important results have been achieved. Septin and other gene family members are involved in the process of microsporidian adhesion and in...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/569
CPCG01N33/68G01N33/56905G01N2333/44
Inventor 马强党晓群刘方燕潘国庆
Owner CHONGQING THREE GORGES MEDICAL COLLEGE
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