CAJ | 학술논문

利用PCR技术扩增日本血吸虫SjCHGC06822 ORF全长序列，以pET28a(+)为载体构建重组质粒，大肠杆菌表达系统进行表达，His-Ni柱层析纯化rSjCHGC06822/His蛋白后，生物信息学分析其可能结构，Western blot分析其抗原性。利用重组蛋白免疫BALB/c小鼠评估其免疫效果，ELISA分析血清抗体亚型变化。结果显示，获得日本血吸虫SjCHGC06822的完整ORF序列591 bp，编码192个氨基酸，序列aa37~aa72含一个EF-hand手性域结构，无信号肽及跨膜结构。获得的SjCHGC06822/His重组蛋白具有较好的抗原性，在2次独立BALB/c小鼠实验中，与PBS对照组相比，SjCHGC06822/His蛋白免疫组诱导小鼠获得30.36%、40.26%减虫率和30.14%、12.28%肝脏减卵率，血清IgG抗体免疫及感染后持续增高，IgG2a和IgG1水平随免疫次数增高，感染后出现下降，但IgG2a/IgG1（<1）水平持续增加。本研究成功克隆了SjCHGC06822基因，并成功表达纯化了rSjCHGC06822/His蛋白，且免疫原性和反应原性良好。SjCHGC06822蛋白免疫可以诱导BALB/c小鼠产生一定的保护力，诱导宿主的免疫应答更偏向于Th1型。
이용PCR기술확증일본혈흡충SjCHGC06822 ORF전장서렬，이pET28a(+)위재체구건중조질립，대장간균표체계통진행표체，His-Ni주층석순화rSjCHGC06822/His단백후，생물신식학분석기가능결구，Western blot분석기항원성。이용중조단백면역BALB/c소서평고기면역효과，ELISA분석혈청항체아형변화。결과현시，획득일본혈흡충SjCHGC06822적완정ORF서렬591 bp，편마192개안기산，서렬aa37~aa72함일개EF-hand수성역결구，무신호태급과막결구。획득적SjCHGC06822/His중조단백구유교호적항원성，재2차독립BALB/c소서실험중，여PBS대조조상비，SjCHGC06822/His단백면역조유도소서획득30.36%、40.26%감충솔화30.14%、12.28%간장감란솔，혈청IgG항체면역급감염후지속증고，IgG2a화IgG1수평수면역차수증고，감염후출현하강，단IgG2a/IgG1（<1）수평지속증가。본연구성공극륭료SjCHGC06822기인，병성공표체순화료rSjCHGC06822/His단백，차면역원성화반응원성량호。SjCHGC06822단백면역가이유도BALB/c소서산생일정적보호력，유도숙주적면역응답경편향우Th1형。
The present study aimed at obtaining the recombinant SjCHGC06822 protein by using prokaryotic expression system and evaluating the immune response in BALB/c mice. A full-length ORF encoding SjCHGC06822 of S.japonicum was amplified in PCR. The obtained cDNA was subcloned into pET28a(+) vector that was then transformed into BL21(2E). The recombinant protein was purified using His-Ni resin. Bioinformatics analysis showed that the full-length ORF was 591 bp, encoding 192 amino acids. The SjCHGC06822 protein had an EF-hand domain without signal peptide and transmembrane peptide. The recombinant protein was visualized as a band at molecular mass of 23 kDa in Western blot. BALB/c mice were immunized with the recombinant protein and antibody responses were detected in ELISA. The immunized mice along with non-immunized controls were challenged with S.japonicum and worm reduction rate and liver egg reduction were recorded. In two separate animal experiments, worm reduction rates of the immunized mice were 30.36%and 40.26%and liver egg reductions were 30.14%and 12.28%. The specific IgG, IgG2a and IgG1 were detected using ELISA. The antibody responses gradually increased post immunization and subsequent challenge. However, IgG2a and IgG1 levels increased post immunization but decreased post challenge while the ratio of IgG2a/IgG1 (<1) was climbing. In conclusion, the recombinant SjCHGC06822 protein can induce good immunogenicity.