Optimization Of Prokaryotic Expression Of 27kDa Cysteine Protease Gene Of Spirometra Erinacei And The Research On Tissues Localization

Objective: To clone and express 27 kDa cysteine protease(CP)gene of Spirometra erinacei and to optimize the prokaryotic expression conditions.Applying immunofluorescence technique to detect the 27kDa-CP distribution in different tissues of adult,pleroceroid of Spirometra erinacei.Methods:(1)A total of RNA was extracted from the plerocercoids and reversely transcribed into cDNA.The 27kDa-CP gene was amplified by PCR and cloned into pET-28a(+)vector according to NCBI released the pleroceroid 27 kDa cysteine protease gene sequence(27kDa-CP D63670).Constructing the recombinant plasmid pET-28a(+)-27kDa-CP,The recombinant plasmid was transformed into Transetta(DE3)and followed by expression of the protein induced by different concentrations isopropyl-β-D-thiogal actopyranoside(IPTG),culture time and temperture.The target protein was purified by Ni2+ affinity chromatography,and analyzed by SDS-PAGE and Western Blotting.(2)Using purified recombinant protein immuned mouse to prepare the polyclonal antibodies(primary antibody).Finally,the 27kDa-CP were immunofluorescence located in adult and pleroceroid tissues using the Anti-mouse IgG/FITC as secondary antibody.Results:(1)The recombinant plasmid of PET-28a(+)-27kDa-CP was successfully expressed in Transetta(DE3)after induction by IPTG.The optimum conditions were as follows: the culture temperature was 30℃,the concentration of IPTG was 0.3mmol/L,and the culture time was 18 h.Recombinant protein mainly existed in the form of inclusion body,and the purified concentration was 0.2 mg/mL.(2)The purified recombinant protein was detected by Western Blotting.The results showed that its relative molecular mass(Mr)was about 35 kD,which was consistent with the expectancy,and could be recognized by immune mouse serum with recombinant protein.(3)Immunofluorescence localization showed that 27kDa-CP concentrated abundantly on the tegument and excretory ducts of pleroceroid.In adult,it also distributed strongly in egg shell of proglottid uterus,followed by excretory ducts and tegument.Conclusion:(1)The prokaryotic expression system of 27kDa-CP gene of Spirometra erinacei was successfully constructed and the expression conditions were optimized.The recombinant protein there was a higher expression level using the optimum conditions in Transetta(DE3).(2)It is suggested that 27kDa-CP may be involved in the invasion,migration,nutrient absorption,excretion,immune evasion,growth and development,reproduction processes according to tissues localization results by immunofluorescence technology.