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Method for analyzing expression level of calcium-sensing receptor (CaSR) after RV and NSP4 infection

An analysis method and expression level technology, applied in the direction of analysis materials, biomaterial analysis, biochemical equipment and methods, etc., can solve the problem of little understanding of the influx mechanism of Ca2+ ions

Inactive Publication Date: 2018-09-07
东莞市第三人民医院 +2
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Problems solved by technology

[0005] intracellular Ca 2+ Dramatic changes in ion stability can be used as important markers of RV infection. The entire process of RV infection, including cell invasion, transcriptional activation, morphogenesis, cell lysis, release of virulent particles, and subsequent protein behaviors, all depend on Ca 2+ completed [16], but the influx mechanism of Ca2+ ions is still poorly understood

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Embodiment Construction

[0022] The present invention will be described below in conjunction with specific embodiments.

[0023] A method for analyzing the expression level of CaSR after RV and NSP4 infection, characterized in that it includes the following steps, specifically:

[0024] a. Cultivation and amplification of virus strains: the cultivation and amplification of RV-Wa strain and SA-11 virus strain are all carried out on monkey embryonic kidney cells; wherein, the monkey embryonic kidney cells inoculated with RV-Wa strain need to be After activation by trypsin, when the cells show serious cytopathic changes, store the cell bottle in the -20°C refrigerator overnight, dissolve at 4°C the next day, and freeze and thaw repeatedly three times, collect the cell freeze-thaw solution, centrifuge, aliquot, and store in the refrigerator for later use ;

[0025] b. Preparation of GST-NSP4 T fusion protein;

[0026] c. Construction of RV diarrhea model and NSP4 diarrhea model: animals were inoculated...

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Abstract

The invention discloses a method for analyzing the expression level of a calcium-sensing receptor (CaSR) after RV and NSP4 infection. The method comprises the following steps: a. culturing and amplifying virus strains: culturing and amplifying an RV-Wa strain and an SA11 strain on monkey embryonic kidney cells; b. preparing GST-NSP4T fusion protein; c. constructing an RV diarrhea model and an NSP4diarrhea model; d, observing body weight, physical signs and diarrhea degree, and determining fecal RV content; e. taking an animal material; f. detecting related indicators: taking paraffin sectionsof all intestinal segments at all time points after RV infection for HE staining, observing morphological changes by using a light microscope, detecting intestinal epithelial cell apoptosis in situ by using a TUNEL fluorescence development process. The method provided by the invention can effectively reveal the expression state of the CaSR in RV and NSP4 infection, thus clarifying the correlationbetween the RV infection and the CaSR expression change.

Description

technical field [0001] The invention relates to the technical field of research on rotavirus infectious diarrhea, in particular to a method for analyzing the expression level of CaSR after RV and NSP4 infection. Background technique [0002] Rotavirus infectious diarrhea presents a high morbidity rate and high mortality rate, and there is no specific drug. Rotavirus (RV) is the main pathogen of rotavirus infectious diarrhea. About 110 million newborns around the world suffer from severe secretory diarrhea every year due to RV infection, and about 50 million children under the age of 5 are infected with RV. Infected and died of dehydration. Vaccination still seems to be the most effective way to prevent RV infection, however, due to the variability of RV strains, problems related to the efficacy and safety of vaccines limit its promotion. At present, oral rehydration solution recommended by WHO is still used in the clinical treatment of acute rotavirus diarrhea to relieve s...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12Q1/6883G01N33/68
CPCC12Q1/6883C12Q2600/158G01N33/5044G01N33/6893G01N2800/26
Inventor 蒲荣黄浩海
Owner 东莞市第三人民医院
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