Construction Of Eukaryotic Expression Vector Of Tubulin ?-2b Chain Mutant And Its Application To Microtubule Polymerization

The microtubules are composed of alternating ?-tubulin and ?-tubulin.The tubulin beta-2B chain(TUBB2B)is a subtype of ?-tubulin,which is the main component of microtubules.Mutations in the tubulin gene TUBB2 B alter the normal function and structure of microtubules.The mutation of asparagine to serine was found at position 247 of TUBB2 B caused damage and abnormal development of mouse cerebral cortex.This paper analyzes the effect of TUBB2 B mutant on microtubule polymerization.In this experiment,the total RNA of C57bl/6j mouse was used as a template,and the cDNA was reverse transcribed.The TUBB2 B cloning vector was constructed by ligation with T vector,and the TUBB2B(N247S)cloning vector was constructed by point mutation technique.The TUBB2B-p3-flag-cmv-10 eukaryotic expression vector was constructed by DNA restriction enzyme digestion,and the eukaryotic recombinant expression vector and empty vector were transfected into mouse fibroblasts under the guidance of transfection reagent.In the fibroblast L929,a blank group and a control group were set up.and tubulin was extracted from different groups of cells,and the expression of ?-tubulin was analyzed by Western-blot.The mutant protein was expressed,collected and purified by magnetic separation techniques.In order to study the effect of the mutant protein on the microtubule skeleton,the purified mutant protein was analyzed in vitro,and the results were as follows:1.The constructed TUBB2 B and TUBB2B(N247S)cloning vectors were confirmed to be correctly constructed by PCR and DNA identification.2.The target fragment was successfully ligated with the eukaryotic expression vector,and the TUBB2B(N247S)-p3-flag-cmv-10 eukaryotic recombinant expression vector was successfully constructed by PCR,double enzyme digestion and DNA sequencing.3.Successfully transfected L929 cells and successfully expressed mutant proteins.After optimization and induction of expression conditions,it was determined that the ratio of the best target gene to transfection reagent was 8:1,and the TUBB2 Bgene was achieved.Efficient expression in L929.4.The transfection group was compared with the blank group and the control group.The free ?-tubulin was significantly higher than the control group and the blank group(P values were 0.0001 and 0.0004,respectively),and the aggregated ?-tubulin was significantly lower than the control group.In addition,blank groups(P values are 0.0004 and 0.0036,respectively).5.Magnetic separation technology can obtain higher concentration of target protein.6.TUBB2B(N247S)mutant protein has a certain inhibitory effect on the in vitro polymerization of tubulin.