日本血吸虫硫氧还蛋白编码基因的克隆表达及其免疫原性研究

Studies on cloning and expression of the gene encoding Schistosoma japonicum thioredoxin and its immunogenicity

Schistosomiasis japonica remains a major public health problem in China despite the remarkable successes in schistosomiasis control achieved over the past four decades. The present strategy to control schistosomiasis relies primarily on diagnosis and targeted chemotherapy supplemented with Oncomelania control through mollusciciding and environmental modification. Praziquantel is an effective drug currently available for human treatment, but it cannot prevent re-infection, and the development of drug resistance by schistosomes and existence of numerous livestock for continued transmission of this parasitic disease to human being have mainly been in concerns. It seems that the most feasible long-term solution to the problem of schistosomiasis at present is a protective vaccine. Considerable efforts have been aimed at the identification of relevant schistosome antigens that may induce protective immune response. Many putative vaccine antigens for schistosome have been identified during the past decades, such as GST, TPI, paramyosin, myosin, membrane-associated antigen （23 kDa） FABP and so on. Unfortunately, only partial protection was reached with any of these candidate antigen formulations. Actually it is very common that the vaccines currently being tested in many laboratories have only shown limited efficacy in animal trials. There remains a urgent need for continuous search for new vaccine candidates. Thioredoxins （Trx） are a class of small 12 kDa redox proteins known to be present in all eukaryotic and prokaryotic organisms. It acts as electron carriers necessary for the catalytic cycles of biosynthetic enzymes, such as ribonucleotide reductases, methionine sulfoxide reductases and sulfate reductases. It generally protects cytosolic proteins from aggregation or inactivation via oxidative formation of intra- or inter-molecular disulfides and is able to reduce H2O2 and scavenge free radicals. It is associated with regulation of apoptosis, immunomodulation, etc. According to the results from our proteomics analysis of S. japonicum （Chinese strain）, SjcTrx is one of the abundantly expressed proteins. To evaluate its immunogenicity, the gene encoding SjcTrx was amplified by RT-PCR and cloned into pcDNA3 vector. The result showed that pcDNA3-SjcTrx induced certain protective efficacy in mice against challenging infection. The gene encoding SjcTrx was also cloned and expressed in E. coli system. The purified recombinant protein, reSjcTrx, was investigated in C57BL/6 mice for the protective immunity efficacy. HT5”HSTHZ1 Construction of DNA vaccine of Schistosoma japonicum thioredoxin and observation of its protective immunity in mice HT5”SSAccording to the cDNA sequence of S. japonicum （Philippine strain） thioredoxin, a couple of primers were designed with the BamHⅠ restriction endonuclease site introduced in forward primer with ATG as start codon and EcoRⅠ in reverse primer with TGA as termination codon. The gene encoding S. japonicum （Chinese strain） thioredoxin （SjcTrx） was amplified by RT-PCR and cloned into the eukaryotic expression vector of pcDNA3. The recombinant plasmids were then transformed into competent E. coli JM109 and identified by endonucleases digestion, PCR, agarose gel electrophoresis and sequencing. The results showed that the RT-PCR product was about 334 bp judged by agarose gel electrophoresis. The same fragments were obtained by restriction enzyme digestion with the recombinant plasmid and PCR with the plasmid DNA as a template. The recombinant plasmid, designated pcDNA3-SjcTrx, was sequenced and shown to be 97% and 43% identical in deduced amino acid sequences to that of S. japonicum （Philippine strain） and S. mansoni thioredoxin, respectively. Thirty C57BL/6 female mice were divided into three groups, 10 mice each group: pcDNA3-SjcTrx group, pcDNA3 control group and challenging group. In pcDNA3-SjcTrx group, each mouse was immunized intramuscularly with 100 μg pcDNA3-SjcTrx at week 0, 2 and 4. In pcDNA3 control group and challenging control group, each mouse was immunized with 100 μg pcDNA3 palsmid DNA and saline as the same immunized schedule. Three weeks after final injection, each mouse was challenged with （30±1） cercariae of S. japonicum（Chinese strain）. All mice were sacrificed and the worms were collected by perfusion six weeks after challenge. The numbers of recovered worms and eggs from hepatic tissue of mice were counted. Sera samples were collected from mice before immunization, before challenge and before sacrifice, and were analysed with ELISA assay to detect specific anti-SjcTrx antibodies. Results showed the that specific antibodies developed in mice against SjcTrx. Comparing with challenging control group, the worm reduction rate and egg reduction rate in liver tissues of pcDNA3-SjcTrx group were 45.7% and 41.4%, respectively （P<0.05 ）. Another 6 C57BL/6 mice were immunized with pcDNA3-SjcTrx as above （i.m.）, and the fresh muscle tissues of injection area were separated for frozen section at 24, 48 and 72 h post-immunization. The result of immunoenzymatic staining technique （IEST） showed that pcDNA3-SjcTrx could express in the local tissues. The results suggested that the DNA vaccine of Schistosoma japonicum （Chinese strain） thioredoxin had good immunogenicity and induced certain immunoprotective capacities against schistosomiasis japonica in mice. Further researchs should be preformed for the DNA vaccine. HT5”HSTHZ2 Cloning, expression of the gene encoding Schistosoma japonicum thioredoxin and its protective efficacy in mice HT5”SSA couple of primers were designed with the BamHⅠ restriction endonuclease site introduced in forward primer with ATG as start codon and SalⅠ in reverse primer with TAA as termination codon. Total RNA was isolated from adult worms of S. japonicum and the SjcTrx gene was amplified by reverse transcriptase-polymerase chain reaction （RT-PCR）. The PCR products and the pET28a plasmids were digested by both restriction endonucleases BamHⅠ and SalⅠ. The target DNA fragments were purified and cloned properly into the prokaryotic expression vector pET28a. After identification by endonucleases digestion, PCR and sequencing, the recombinant plasmid pET28a-SjcTrx was transformed into competent E. coli BL21 and expressed in the presence of IPTG. The RT-PCR product was about 334 bp judged by agarose gel electrophoresis. The same fragments were obtained by restriction enzyme digestion from the recombinant pET28a-SjcTrx plasmid and PCR with the plasmid as a template. The recombinant pET28a-SjcTrx plasmid DNA was sequenced and shown to be 97% and 43% identical in deduced amino acid sequence compared with thioredoxin of S. japonicum （Philippine strain） and S. mansoni, respectively. Induction of the gene expression in E. coli BL21 showed a constant level of recombinant protein production. The results of SDS-PAGE and Western blot revealed that the molecular weight of expressed protein was around 14 kDa that could be recognized by sera from rabbits infected with S. japonicum and from mice immunized with reSjcTrx. Thirty six-week-old C57BL/6 female mice were divided into 3 groups, 10 mice each: reSjcTrx with Montanide ISA720 adjuvant, adjuvant control and challenging control. Mice were vaccinated subcutaneously at week 0, 2, 4 with reSjcTrx emulsified in Montanide ISA720 adjuvant. In adjuvant control, each mouse was injected three times with Montanide ISA720 and saline. In challenging control group, mice were given no injection. Three weeks after final injection, each mouse was challenged with （30±1） cercariaes of S. japonicum （Chinese strain）. At the week six after challenge, all mice were sacrificed and perfused. The number of recovered worms and eggs from hepatic tissue of mice were counted. Sera were collected from mice before immunization, before challenge and before sacrifice. High level of specific IgG antibodies was detected by ELISA in the mice immunized with reSjcTrx. The worm reduction rate and egg reduction rate of reSjcTrx immunization group were 22.8% and 29.5 % respectively, compared with control group（P<0.05）. The results suggested that the recombinant antigen could induce certain immunoprotective capacities against schistosomiasis japonica in mice.

Schistosomiasis japonica remains a major public health problem in China despite the remarkable successes in schistosomiasis control achieved over the past four decades, The present strategy to control schistosomiasis relies primarily on diagnosis and targeted chemotherapy supplemented with Oncomelania control through mollusciciding and environmental modification. Praziquantd is an effective drug currently available for human treatment, but it cannot prevent re-infection, and the development of drug resistance by schistosomes and existence of numerous livestock for continued transmission of this parasitic disease to human being have mainly been in concerns.