Studies On Molecular Cloning,Expression And Expression Pattern Of The CDNA Encoding 27kDa Cysteine Protease Of Spirometra Erinacei

Objective: To analyze the molecular biological characteristics and observe the time and space expression patterns of the cDNA encoding 27 kDa cysteine protease(CP) of S.erinacei. Methods: 1.Sequence analysis, clone, and expression of the encoding cDNA of CP:(1)Bioinformatics tools, such as Ex PaSy and NCBI were used to predict and analyze the structure and biological function of deduced amino acid of CP.(2)SEP-CP fragment was inserted into the prokaryotic expression vector pET-28a(+), and then pET-28a(+)-SEP-CP was transformed into E. Coli Transetta/DE3.The recombinant plasmid was expressed under the induction of IPTG.SDS-PAGE was used to analyze expression product. 2.Detection of the space-time expression pattern of CP:(1)The plerocercoids were taken from the snakes(Elaphe carinate) caught in Anshun, Guizhou. A total of 64 Kunming mice(20-25 g) were equally divided into 8 groups, each of them was orally infected with 5 plerocercoids(8 mice/each group). The mice were sacrificed to observe and collect the parasitic plerocercoids after 30 minutes to 14 days.(2)The three dogs were infected with 10 plerocercoids and were sacrificed to observe and collect the parasitic adults after 30 days.(3)Using the 18 S rRNA(18S ribosomal RNA) as reference gene, the expressions of 27 kDa CP gene were detected by real-time quantitative PCR in different worm positions and infective stages of pleroceroid. Results: 1. The results showed that the molecular weight and isoelectric point of predicted CP were 35669.9Da and 5.92 respectively, and 27 kDa CP gene belonged to the Peptidase_C39_like superfamily. 2.SDS-PAGE results showed the expression product was about 35 kDa and mainly existed in the form of an inclusion body.3.The plerocercoids were found to penetrate into the stomach or small intestine wall of mice and appeared in abdominal cavityafter 30 minutes of infection. After 30 minutes to 6 hours, the majority of plerocercoids distributed in gastrointestinal lumina, gastrointestinal walls and abdominal cavities of mice. After1 day of infection, a few plerocercoids migrated to subcutaneous muscle tissue. It was within 7 days that most plerocercoids migrated to subcutaneous muscle tissues. 4.The expressions of 27 kDa CP gene in different positions of adult and pleroceroid were detected, but below the larval stage. The adult expression level in order of size was gravid proglottid>scolex>mature proglottid,while that of pleroceroid was body fragment>head fragment>tail fragment. 27 kDa CP gene of pleroceroid increased 30 minutes after infection, was not infected 2.1 times,and decreased to the lowest 6 h after infection, and then rose to the pesk value at the14 th day.Conclusion: 1.The 27 kDa cysteine protease gene of pleroceroid belonged to the Peptidase_C39_like superfamily. 2. pET-28a(+)-SEP-CP was constructed successfully. 3. The time and space expression patterns of the 27 kDa cysteine protease gene of the pleroceroid and the S.erinacei indicated that cysteine protease may play an important role in the invasion,migration,nutrient absorption,growth and reproduction of the S.erinacei.