Effect of Escherichia coli Cry1Ⅰa protein on BRL-3A cell apoptosis
Objective To express and purify Escherichia coli crystal(Cry)1Ⅰa protein,and study its effect on apoptosis of BRL-3A cells.Methods The coding gene of Escherichia coli Cry1Ⅰa gene was inserted into the pET28aDel expression vector,and the expression vector was transferred into the Escherichia coli BL21(DE3)star strain for the expression and purification of Cry1Ⅰa protein.The hypoxia model of rat BRL-3A cells was established by cobalt chloride treatment,and the cells were divided into blank group(without any treatment),control group(treated with cobalt chloride only),and intervention groups(treated with Cry1Ⅰa protein at concentrations of 0.3,1,3,and 10 μg/mL after cobalt chloride treatment).The apoptosis of BRL-3A cells was detected by flow cytometry,and the expression levels of proteins related to the apoptosis signaling pathway of nuclear factor-κB(NF-κB)(NF-κB and Caspase3)were detected by Western blot.Results The expression vector of Escherichia coli Cry1Ⅰa protein was successfully constructed and transformed into BL21(DE3)star strain.The protein was expressed with a molecular weight of about 81 000 Mr after isopropyl β-D-thiogalactoside induction.The apoptosis rate of cells in the blank group was significantly lower than that in the control group and the intervention groups(F=18.642,P＜0.001).The apoptosis rate showed a downward trend with the increase of intervention concentration.The expression level of NF-κB protein in the NF-κB signaling pathway in the blank group was significantly lower than that in the control group and the intervention groups(F=20.518,P＜0.001).The expression level of Caspase3 protein in the NF-κB signaling pathway in the blank group was significantly lower than that in the control group and the intervention groups(F=29.734,P＜0.001).The expression level showed a downward trend with the increase of intervention concentration.Conclusion Escherichia coli Cry1Ⅰa protein could reduce the apoptosis of hypoxic BRL-3A cells,and its mechanism might be related to the inhibition of NF-κB signaling pathway.
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