| Schistosomiasis is a zoonotic parasitic disease caused by infection of cercariae,which causes serious pathological damage to the host.Schistosomiasis is one of the six key tropical diseases identified by WHO.Due to the complexity of the multistage development and life cycle of schistosome,the mechanism of immune response and escape of the host induced by the parasite are not very clear,which restricts the research on schistosomiasis vaccine with good protective effect.Therefore,finding more effective vaccine candidate molecules may become an important way to prevent and control schistosomiasis.Transmembrane proteins are located at both ends of the biological membrane and penetrate between the cell and the outside world.They have many important functions such as mediating signal transduction,molecular transportation,and energy conversion.The transmembrane protein located on the cover of schistosome is in direct contact with the host’s immune system.Therefore,transmembrane proteins may be an important molecule for information exchange between the host and the worm.It is also necessary to develop functional studies of transmembrane proteins for the development of S.japonicum vaccine.In this paper,the function of transmembrane protein TMEM66 P in the parasitic and reproductive development of schistosome was preliminarily explored,including the following three aspects:(1)Cloning,expression and bioinformatics analysis of cDNA encoding S.japonicum TMEM66PWe designed the primers according to the sequence of S.japonicum TMEM66 P,and the c DNA fragment of this sequence was amplified by PCR.Sequence analysis showed that the amplified cDNA fragment included an open reading frame of 1008 bp,and the predicted molecular weight of the encoded protein was 37.93 kD.Bioinformatics analysis showed that the homology of TMEM66 P was 67.79% with schistosoma mansoni and 44.54% with mice.In addition,the prokaryotic recombinant protein pET32a(+)-TMEM66 P was successfully constructed,expressed and purified.(2)Expression of TMEM66 P in Different Developmental Stages and Gender of S.japonicumThe transcription level of TMEM66 P in different developmental stages and genders of S.japonicum was detected by RT-PCR.The results showed that the TMEM66 P was expressed at different developmental stages of S.japonicum.The expression level in S.japonicum eggs and the worm infected mice at 35 days were significantly higher than that at 14 days and 21 days,followed by that at 28 days.In addition,the expression of TMEM66 P in males was higher than that of females in 28 days worms infected with mice,and the expression of TMEM66 P in males was significantly higher than that in females in 35 days infected.The above results indicate that TMEM66 P has differential expression in different developmental stages and different genders,suggesting that this gene may play an important role in the reproductive spawning and growth of S.japonicum.(3)Preliminary study on the function of S.japonicum TMEM66PRNAi technology was used to interfere the expression of TMEM66 P in vitro,and the screening results were analyzed at the transcription level to select the small RNA molecule with the best interference effect(siRNA-664).RT-PCR was used to detect the eggshell protein gene of the interfering worms,and it was found that the eggshell protein gene AY222895.1,AY814244.1,M59318.1 and D32205.1 expression levels were significantly reduced.The CellTiter-Lumi ? method was used to detect the activity of worm cells after interference,the vitality of worm cells decreased significantly;A statistical analysis of the egg production of the worm revealed that the egg production decreased significantly after interference;As the interference time prolonged,the worm’s vitality gradually decreased.It showed that the decrease of TMEM66 P expression level had an effect on female egg laying and worm activity,suggesting that TMEM66 P may play an important regulatory role in the reproductive development of S.japonicum. |
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