Cloning Expression And Functional Analysis Of The Coding Region Of Pig BLM Helicase Gene

BLM helicase,a member of the RecQ helicase family,plays a key role in cell DNA metabolism including replication,recombination,repair,maintaining stable in chromosome terminal and so on.Human BLM gene mutation will reduce the activity of BLM helicase and block the DNA repair process leading to chromosome instability,which can lead to Bloom syndrome?BS?.BS is a rare recessive autosomal genetic disorder that predisposes patients to various types of cancer.However,there are few reports on the correlation between BLM gene and disease in livestock and poultry.In this experiment,Cong jiang xiang pig was selected as the research object,and PCR amplification,molecular cloning,prokaryotic expression,affinity chromatography,fluorescence polarization and other methods and technologies were used to study the structure and function of the BLM gene in pigs,so as to provide scientific basis and new ideas for the occurrence,development,prevention and control of major diseases in pigs.The results had been revealed in follows:?1?The qRT-PCR results showed that compared with the heart tissue,the expression of the BLM gene from pig was detected in all tissues,with the highest relative expression in testis tissue,followed by the tissues of lung,liver,spleen,heart,kidney,small intestine and large intestine.The expression levels in the testis and lung tissue were higher than other tissues,and the difference was extremely significant?P<0.01?.The expression of BLM gene in lung tissue of pig porcine reproductive and respiratory syndrome,liver tissue of chicken leukemia and liver tissue of quail duck was higher than that in normal tissue,and both were significant differences?P<0.05?.The difference indicates that changes in BLM helicase may also lead to animal diseases.?2?The sequences of BLM gene coding regions from Cong jiang xiang pig were successfully cloned.There were 4 base mutations in the BLM gene sequence compared with those in the GenBank.The mutation changed leucine at 1158 to proline,glycine at 1362 to serine,and the rest to nonsense mutations.Amino acid sequence comparison showed that the amino acid sequence of the pig BLM gene was the highest homology of the cow?83.7%?.The phylogenetic tree analysis showed that the porcine BLM helicase had four genetic domains of DEXDc,HELICc,RQC and HEDC;Phylogenetic tree analysis showed that pig BLM is most closely related to cow BLM.?3?The prokaryotic expression vector pET-32a-BLM^673-1301was successfully constructed.The sequencing results showed that the sequence length of pig BLM⁶73-130173-1301 gene was 1884bp and there was no base mutation.The physical and chemical properties of the protein showed that pig BLM^673-1301contained 628 amino acids with a molecular weight of 71.14 kD and a theoretical isoelectric point of 8.70.?4?The recombinant protein of pig BLM^673-1301helicase was successfully induced by pET-32a-BLM^673-1301in vitro.Pig BLM^673-1301helicase was mainly expressed as inclusion body.Orthogonal experiment design was used to optimize the pig BLM^673-1301helicase soluble expression,the results showed that 20?,8 h,1 mmol/L IPTG,220 rpm for optimal combination of pig BLM^673-1301helicase soluble expression.Anova showed that temperature,time,concentration and rotation speed all had extremely significant influence on the expression of pig BLM^673-1301helicase,among them,temperature has the greatest influence.Pig BLM^673-1301helicase was purified by Ni⁺affinity chromatography.The optimal concentration of imazole was 100 mol/L and elution was 500 mmol/L.The purified target protein concentration was 2.7 g/L.The protein was identified and purified by westernblot as Pig BLM^673-1301helicase.?5?The biological activity of pig BLM^673-1301helicase was detected by fluorescence polarimeter.The results showed that the purified pig BLM^673-1301helicase had binding and chain deactivation activities.