Construction Of Recombinant Lentiviral Vector For Goat ATF6 Gene Interference And Its Effects On Apoptosis Of Goat Trophoblastic Cells

Activating transcription factor 6(ATF6)is a type II ER transmembrane protein and it’s a key regulator of the unfolded protein response(UPR).After cleaved as p50ATF6,which is able to migrates to the nucleus to activate ER-associated degradation(ERAD)gene expression,also can heterodimerize with XBP1 protein,promoting unfolded or misfolded protein to folding and degradation,helps restore ER protein folding homeostasis and alleviates ER stress.Various tissues,organs and cells have ATF6 expression,and goat trophoblast cells(GTCs)is one of them.Trophoblast cells is critical to the development of the process of pregnancy,but apoptosis of GTCs effect on the pregnancy is not clear enough.In this study,ATF6 was taken as the entry point to lay a foundation for studying the effect of ATF6 on apoptosis of GTCs.This experiments mainly used the PCR,lentiviral vector,production of lentivirus,qRT-PCR,Western Blotting and Flow CytoMeter(FCM),constructed the recombinant lentiviral vector for goat ATF6 gene interference,and the effect of inhibiting ATF6 expression on GTCs apoptosis by ER stress pathyway was preliminarily analyzed.Major results include:1.Designed a pair of primers and clone the coding sequence(CDS)of goat ATF6 by PCR,and the CDS we finded had highest homology with Capra hircus(100%),Ovis aries(99%)and Pantholops hodgsonii(96%).Analysis of the amino acid sequence,we know the molecular weight is 157996.66 u,the theoretical p I is 4.92,the instability index(II)is computed to be 47.44,the aliphatic index is 28.92,the grand average of hydropathicity(GRAVY)is 0.883;2.Designed and constructed three pairs of recombinant lentiviral vectors for the CDS of goat ATF6 gene(pCD513B-U6-shRNA-ATF6-CDS-1,pCD513B-U6-shRNA-ATF6-CDS-2,pCD513B-U6-shRNA-ATF6-CDS-3);3.Used HEK293 T cells to produced recombinant lentiviruses,then through endometrial epithelium cells(EECs)infected with recombinant lentiviruses to analysis of mRNA and protein expression level of ATF6,filter out the ATF6 expression level reduced to less than 70% after delivery of pCD513B-U6-shRNA-ATF6-CDS-2 lentiviral vector,and used it to infected GTCs,get shATF6-GTC for use in subsequent tests;4.After Tm induction,shATF6-GTC and shNC-GTC both showed early apoptosis,and the early apoptosis rate of shATF6-GTC was significantly lower than sh NC-GTC,indicating that the inhibition of ATF6 expression may promote the survival of GTCs.