Effect Of PLK1 Gene Expression On The Development And Differentiation Of Echinococcus Granulosus

Objective:(1)The physicochemica properties and biological functions of PLK1 protein were analyzed by bioinformatics.(2)The protoscolex of Echinococcus granulosus and Echinococcus multilocularis were obtained from infected animals,and the effects of protoscolex evagination and adult culture conditions on the development in vitro were explored.(3)The PSC was cultured in vitro to develop towards adults,and the expression difference and localization of PLK1 in different developmental stages of parasites were explored.(4)To explore the influence of the deletion of PLK1 gene on the development and differentiation of PSC.(5)To explore the expression and location of PLK1 in the liver tissue samples of mice infected with E.g and E.m and patients infected with AE,CE,so as to lay a foundation for clinical molecular diagnosis of hydatidosis and targeted drug therapy.Methods:(1)By searching the genome database of Echinococcus granulosus in NCBI website,the c DNA sequence of PLK1 was obtained,and specific primers were designed.The total RNA of Echinococcus granulosus protoscolex was extracted,and the PLK1 gene was amplified by routine PCR using this template.The PCR product was subjected to gel electrophoresis,and the target fragment was cut and sequenced and then analyzed by bioinformatics.(2)PSC was obtained in vitro and cultured in vitro.The total RNA and total protein of parasites at different developmental stages were extracted.The difference of expression of PLK1 from PSC to adult development was analyzed by RT-q PCR and Western Blotting techniques.(3)PSC was cultured in vitro for 56 days,and samples were collected at different stages.The worms were fixed with 10%agarose solution heated to 65 ℃,paraffin sections were prepared for HE staining,and the expression of PLK1 in the worms was localized by immunohistochemistry.(4)The head and neck segments of the adult worm and the worm body were separated under the light microscope,and the samples were collected.The expression of PLK1 gene m RNA in different parts of the worm body was analyzed by RT-q PCR.(5)The expression of PLK1 was interfered in vitro by electroporation of siRNA transfection into PSC.The survival rate of PSC was counted after three days of PSC culture in vitro,and samples were collected to calculate the expression level of PLK1 gene after different siRNA sequence interference by fluorescence quantitative PCR.(6)The liver tissue samples of mice infected with E.g and E.m for 6 months and AE and CE patients were specifically identified,and the expression and localization of PLK1 monoclonal antibody in the liver tissue of the host infected with hydatidosis were explored by immunohistochemistry.Results:(1)The cloned PLK1 gene sequence is 98.6% similar to the registered transmembrane protein of Echinococcus granulosus in Gene Bank.PLK1gene Encodes 1466 amino acids.The theoretical isoelectric point is 7.33,and the instability index is 36.34,which is an stable protein.The fat coefficient is 106.46,and the average value of hydrophilicity and hydrophobicity is 0.958, which is hydrophobic soluble protein.The protein encoded by PLK1 contain four transmembrane regions and 11 dominant B cell epitopes,which belong to transmembrane protein;(2)Short time pure water stimulation(2×20s) significantly increased the evagination of E.g and E.m protoscolex.However, the medium containing fetal bovine serum(FBS)made 92% of protoscolex invaginate(inhibiting protoscolex evagination).In the serum RPMI-1640 medium with dog bile,the evaginated proscolex maintained 80.5% of the evagination rate after three days of pre-culture,and developed towards adult worms.The dog serum gel base maintained 79.8% of the evaginatd protoscolex and developed into adults,which was higher than the fetal bovine serum gel base.The rapidly developing worms had 3-4 progloids after 56 days’ culture.E. granulosus worms were longer and wider than E.multilocularis after 5 weeks of in vitro culture;(3)PLK1 gene was highly expressed in the germinal layer of E.g cyst,and the expression of PLK1 gene in the adult stage was higher than that in other developmental stages(P<0.05).PLK1 is expressed at the bottom of the developing protoscolex sucker,and it is highly expressed in the head and neck segment region of the worm during the adult development stage.It is known that this region is the only region where the adult segment proliferates.(4)Immunohistochemical results showed that PLK1 polyclonal antibody showed specific positive recognition in the liver lesions of E.g and E.m infected mice,and the positive area was mainly distributed in the contact surface between the host liver tissue and the cyts;The liver tissues of AE and CE patients were identified.The normal tissues around the liver of the patients had no reaction.The pathological tissues of patients with AE and CE infection showed specific positive recognition in liver lesions.The positive areas were mainly distributed in the central area and the necrotic area of the inflammatory zone around the lesion.(5)The survival rates of E.g PSC transfected with siRNA-PLK1-162,siRNA-PLK1-1100 and siRNA-PLK1-1798 were(34.3 ± 4.87)%,(25.4 ±2.98)% and(74.6 ± 4.54)% respectively,and the relative expression of m RNA was(0.46 ± 0.023)(0.26 ± 0.019)and(0.84 ± 0.027),in which the relative expression of siRNA-PLK-1100 sequence m RNA was significantly different from that of the unrelated sequence control group(0.95 ±0.032)and the blank control group(0.98 ± 0.012)(P<0.05),so the best interference sequence was siRNA-PLK-1100.Conclusion:(1)The protein encoded by the PLK1 gene of E.g contains three transmembrane domains,PLK1 protein is stable protein,belongs to hydrophobic soluble protein,contains 11 dominant B cell epitopes,which can be used as a candidate epitopes for subunit vaccines.(2)An in vitro culture model of Echinococcus granulosus and Echinococcus multilocularis PSC developing towards adults was successfully established.Four segments worms were obtained from the worms cultured in vitro for 56 days;Stimulating PSC evagination with pure water for a short time and maintaining PSC evagination,are essential for the development of E.g and E.m in vitro;Dog serum solid pad and dog bile are indispensable factors for the long-term development of 2 types of Echinococcus.(3)PLK1 is obviously located at the bottom of the scolex of PSC and in the germinal layer cells of the growth area of the adult body,which indicats that PLK1 is a highly expressed gene in the stem cell region during the development and differentiation of E.g adult worms.(4)PLK1specifically recognizes the liver tissues of mice infected with E.g and E.m.It has a specific positive recognition for the pathological tissues of AE and CE patients,and can be used as recombinant immunodiagnostic antibody for hydatidosis.(5)By interfering with the expression of PLK1 gene in PSC in vitro,the survival rate of PSC decreased significantly and the expression of m RNA decreased;The optimal interference sequence is siRNA-PLK-1100,so PLK1 can be used as a candidate target for the development of two types of hydatid RNA vaccine and targeted drugs.In conclusion,this study shows that PLK1 plays an important role in the diagnosis of hydatidosis,the development of RNA vaccine and the screening of targeted drugs.