| Schistosomiasis is a kind of zoonosis which seriously threatens human health. Accordingto the statistics from WHO, over243million schistosomiasis patients need to be treated in2011. In China,240,597people are estimated to have the disease till2012. Schistosomiasismainly threatens those in agricultural and fishing industry, in which people are easily exposedto infested water. In addition, the emergence of ecological tour has attracted more and moretravelers to endemic regions, resulting in increased risk of infection. The transfer ofschistosomiasis to new regions may be caused by population floatation and the movement ofrefugees. Moreover, the continuous rise of population and the increasing demand for waterand power may lead to some developmental program and environmental change, which mightincrease disease transmission. The major measure to prevent schistosomiasis is to minimizethe exposure to infested and contaminated water, including the provision of clean water,sufficient hygiene infrastructure and proper health education.Schistosomiasis japonica is the major type of schistosomiasis in China. After60years’efforts, great success has been achieved in the control of schistosomiasis. Among theschistosomiasis-endemic regions in southern China, there are5provinces (cities, autonomousregions) of Shanghai, Guangdong, Guangxi, Fujian and Zhejiang that have interrupted thetransmission of schistosomiasis, where no local infections are detected for successive5years.In addition, transmission control of schistosomiasis has been achieved in the mountainousregions of Sichuan and Yunnan provinces and the marshland and lake regions of JiangsuProvince, where the infection rate of schistosome in residents and domestic animals has beenreduced to less than1%. The prevalence of S. japonicum infection ranges from1%to5%in4provinces of Hunan, Hubei, Jiangxi and Anhui, where the infection rate in bovines was1.04%in2010. Diagnosis, a central part in schistosomiasis control, plays a critical role in detectingpatients, understanding the spatial, temporal and human distribution, identifying thechemotherapeutic targets at individual or mass levels, assessing the chemotherapeutic andpreventive efficacy, as well as the monitoring after transmission control or interruption, whichprovides essential information and scientific evidence for the planning and implementation ofcontrol programs and the assessment of control efficacy. Currently, the infection rates ofschistosome in both humans and bovines are at an extremely low level in China. Theconventional pathogenic techniques are inappropriate for the diagnosis due to low sensitivityand high missing rate. A search for rapid, economic diagnostic methods with high sensitivity,specificity, and compliance has therefore been given a high priority. Many immunological andmolecular biological techniques have been developed for the diagnosis of schistosomiasis. Ascompared to the conventional pathogenic methods, immunological and molecular biologicaltechniques like PCR and LAMP have stronger sensitivity and specificity, low missingdiagnosis rate, and satisfactory compliance, and some molecular biological techniques haveshown early diagnostic values. Immunodiagnosis is mainly dependent on antigen-antibodyresponse. The specific antibody (mainly IgG) may persist in the body of schistosomiasis casesin the endemic regions for a long term, which cannot rapidly disappear even if cured.Therefore, the currently available antibody-based diagnostic methods cannot confirm whether the patients are cured or not (assessment of therapeutic efficacy), resulting in the developmentof over-chemotherapy in the endemic foci. Such a phenomenon causes the huge waste ofpraziquantel and the increase of cost used for schistosomiasis control. In addition, there is aworry about praziquantel resistance following long-term, extensive use of the currently onlyavailable agent used for the treatment of human schistosomiases. Currently, fewer and feweracute schistosomiasis cases are reported in China; therefore, the development of diagnosticmethods with therapeutic assessment is urgently needed.Valosin-containing protein (VCP), a widely distributed ATP enzyme, is one of theATPase superfamily members. It has been proved that VCP is involved in multiple cellularactivities, including mitosis, homotypic fusion, endoplasmic reticulum degradation andubiquitin-dependent protein degradation. It is also reported that VCP is closely related to thecell apoptosis, cancer invasion and metastasis. However, the diagnostic value and function ofVCP in S. japonicum remains unclear. Using molecular cloning, our study obtained rSjVCPthrough gene expression and protein purification, and then we assessed the diagnostic value ofrSjVCP. This study included the following3parts:Part1Gene cloning, prokaryotic expression and purification of SjVCPBased on the protein gene code screened through proteomics, we obtained relevant genesequences by searching in GenBank, and then the bioinformatics software BioXM andDNAMAN were employed to analyze the nucleic acid sequence, digestion site, amino acidsequence, molecular weight of the fusion protein, and isoelectric point. Containing195enzyme cutting sites including XhoI, the DNA sequence of Sj-VCP is2409bp, which A+Taccount for55.09%. Amino acid sequence analysis showed that SjVCP contains179acidicamino acids and119alkaline amino acids. The molecular weight of rSjVCP is90.99KDa andthe isoelectric point (PI) is6.030. RNA was isolated from S. japonicum eggs, reverselytranscribed into cDNA, and amplified for SjVCP gene using PCR assay. The gene was thensubcloned into the prokaryotically expressed vector pET15b. The recombinant plasmid wastransformed into E. coli BL21, and the expression of the target gene was induced withisopropyl-beta-D-thiogalactopyranoside (IPTG). Then the recombinant protein was yieldedusing purification of the inclusion body. Thus, this part of study successfully obtainedrecombination Schistosomiasis japonica valosin-containing protein(rSjVCP).Part2Determination of rSjVCP expression in cercariae, schistosomula, adults (male,female), and eggs of S. japonicumFirstly, RNA was extracted from cercariae, schistosomula, adults (male, female), and eggof S. japonicum, and the DNA present in the RNA samples was digested using DNase. TheRNA samples isolated were then purified, and transcribed into the first chain of cDNA. Theprimers of the target gene SjVCP and the internal reference gene18S were designed to allowcomparative annealing temperatures and amplification products. Taking the1st chain ofcDNA as a template, the corresponding gene fragments in the various life cycles of S.japonicum were amplified using fluorescence RT-PCR assay, and the RNA expression wasquantified using the2-ΔCTmethod. Our findings showed that the highest SjVCP mRNAexpression in the cercariae, and a low expression in the schistosomula, egg, and male andfemale adults. Therefor, the transcription level of SjVCP gene is higher in the early life cycle of S. japonicum than in the late life cycle.Part3Diagnosis value of rSj-VCP for diagnosis of S. japonicum infectionEleven BALB/c mice (5-6weeks old) were numbered A-K, and infected with S.japonicum, of40cercariae each mouse. The blood samples were collected from mice oncebefore infection and1-5weeks post-infection. The sera were detected using, while SEA-basedELISA served as controls. Except number D mouse with positive IgG at the first week, theother10mice were positive for IgG since the second week detected by rSj-VCP-based ELISAassay, and the antibody level showed a decline tendency with the extension of infection time.SEA-based ELISA detected positive sera since week3post-infection, and all mice werepositive for IgG4weeks post-infection. The antibody level appeared an increasing tendencywith the prolongation of the time. Hence, this part of study initially proved that rSjVCP hasthe value of early diagnosis compare with SEA.
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