Prokaryotic Expression And Localization Of Chicken LKB1 And Analysis Of Its Expression In Fatty Liver Hemorrhage Syndrome(FLHS)laying Hens

Liver kinase B1(LKB1),a member of the serine/threonine kinase family,plays an indispensable role in animal organisms.In the current study,chicken LKB1 protein gene was amplified by PCR based on LKB1 primer and c DNA template.We constructed a clone vector of LKB1 target gene using p EASY-T3 vector and cloned a large number of LKB1 target genes.Then,the target gene was inserted into the p ASY-Blunte2 vector to complete the construction of the exogenous recombinant vector.After the expression vector containing the target gene was transferred into BL21(DE3)host cells,the target protein was obviously induced to express by IPTG.The target protein was purified by Ni column,and the rabbit was immunized with the purified,renaturated and concentrated LKB1 target protein to obtain the antibody of the target protein.The antibody titer was detected by enzyme linked immunosorbent assay,and the antibody specificity was detected by Western blot.The expression of LKB1 protein in chicken liver,heart and hypothalamus was mapped by immunofluorescence technique.The LKB1 polyclonal antibody prepared in this study was used to detect the changes of LKB1 protein expression in liver,heart and hypothalamus of FLHS laying hens by Westernblot.Results: Sequencing results showed that the expression vector of chicken LKB1 was successfully constructed,and the prokaryotic expression system could express a large amount of LKB1 recombinant protein.The titer of LKB1 antibody obtained by immunizing rabbits with the above proteins was about1:128000.Western blot assay showed that LKB1 antibody had strong specificity to LKB1 protein in chicken hypothalamus,heart and liver.Immunofluorescence assay showed that LKB1 protein was mainly expressed in the cytoplasm of chicken liver,heart and hypothalamus cells.Westernblot showed that the expression of LKB1 protein in liver,heart and hypothalamus of FLHS laying hens was significantly lower than that in normal group(P<0.05).In this study,the prokaryotic expression system of chicken LKB1 protein was established,and a large number of recombinant LKB1 proteins were obtained,and then polyclonal antibodies against LKB1 protein were prepared.At the same time,the expression of LKB1 protein in the liver,heart and hypothalamus of FLHS chickens was detected by using this antibody.Our study lays a foundation for further study on the role of LKB1 protein in the pathogenesis of chicken FLHS and other diseases.