Cloning,Vector Construction And Functional Verifiction Of Musca Domestica Acetylcholine Receptor Subunits

In the study,using PCR with general primers designed on insect nAChRs subunits,we identified four nAChRs subunits in housefly,the four fragments of musca domestica acetylcholine receptor geneα2-like,α5-like,α6-like andβ3-like were cloned into subunits 1649bp、2340bp、1473bp、1296bp respectively.After sequencing and identification,the homology of the gene sequence with the four subunits Mdα2,Mdα5,Mdα6 and Mdβ3 of Musca domestica nAChR registered by NCBI reached more than 95%.It was proved that the CDS sequence of Musca domestica nAChR subunit was successfully cloned.while theβ2 subunit from rat nAChR gene was synthesized and matched with the sequence registered by NCBI as high as 100%.While successfully cloning the target gene,four nAChR subunits were constructed to the T7 initiation site of the vector PGEM-T by double enzyme digestion,and then the target gene on the vector was transcribed into c RNA with c RNA transcription kit,and injected into Xenopus laevis oocytes at the same concentration asα2/β3,α5/β3,α6/β3,α2/β2,α5/β2 andα6/β2 isoforms for exogenous expression.After 3～5 days cultured,the receptor protein of the 6 groups of nAChR gene interpolymer subtype combination c RNA will be expressed on the surface of oocyte membrane,and then it can be verified whether the cloned gene functional protein can be successfully expressed by Xenopus laevis oocyte by voltage clamp technique.it was found that under the stimulation of acetylcholine gradient concentration（80、40、20、10、5、1μmol/m L）,theα2/β2 subunit interpolymer subtype had functional expression current and there was a positive correlation between current and acetylcholine concentration,the correlation coefficient R² was 0.9207,the activation concentration EC₅₀ was 83.51±4.08μmol/m L,instead the rest 5 groups of subunits interpolymers.However,althoughα2couldn’t be co-expressed with Musca domestica ownβ3 subunit in vitro,theα2/β2 subunit copolymer had functional expression current,indicated that it can be co-expressed with mammalian ratβ2 subunit.After detecting the successful expression of housefly nAChR subunit function in Xenopus laevis cells,the cells were stimulated with target drugs of monosultap and nitenpyram,respectively,and the dose relationship between current and concentration was detected.The EC₅₀of cells successfully expressedα2/β2 subunits was known to be83.51±4.08μmol/ml,according to which recording solution containing 80μmol/m L acetylcholine is configured with concentration gradient（8、4、2、1、0.5μmol/m L）solution of nereistoxin monosultap and neonicotinoid nitenpyram to stimulateα2/β2 subunit interpolymer functional expression cells.It was found that under the stimulation of nitenpyram,the current change was significantly correlated with concentration gradient,with correlation coefficient R² being 0.9507 and EC₅₀being 8.852±3.98μmol/m L,it preliminarily proved that the drug target of neonicotinoid insecticide nitenpyram is Musca domestica nicotinic acetylcholine receptorα2 subunit.