日本血吸虫亮氨酸氨基肽酶和果糖二磷酸醛缩酶重组疫苗对小鼠的保护性免疫效果观察

目的利用重组亮氨酸氨基肽酶(rSjLAP)和果糖二磷酸醛缩酶(rSjFBPA)辅以佐剂免疫BALB/c小鼠,观察基于2-DE和血清蛋白质组筛选的上述二种重组抗原的免疫保护效果;通过福氏佐剂和纳米C60佐剂在增强免疫效果方面的比较,探讨纳米C60佐剂在血吸虫分子疫苗接种中的价值。方法从重组质粒转化的E.coli中诱导表达rSjLAP和rSjFBPA,纯化并检测含量。将6周龄BALB/c小鼠随机分为7组,生理盐水对照组(A组)、rSjLAP+福氏佐剂组(B组)、rSjFBPA+福氏佐剂组(C组)、rSjLAP+rSjFBPA+福氏佐剂组(D组)、rSjLAP+纳米C60佐剂组(E组)、rSjFBPA+纳米C60佐剂组(F组)、rSjLAP+rSjFBPA+纳米C60佐剂组(G组),每组6只。除A组外,B～G组小鼠皮下多点注射rSjLAP或rSjFBPA100μg,共免疫3次,每次间隔2周。用尾蚴感染小鼠6周后,处死小鼠,收集成虫,计算减虫率;收集肝组织虫卵,计算减卵率。结果 B～G组的减虫率分别为:35.59%、33.05%、37.98%、38.99%、43.21%和45.75%,与A组比较,差异均有统计学意义(P<0.05);B～G组的减卵率分别为:40.25%、42.57%、48.88%、41.64%、45.44%和46.88%,与A组比较,差异均有统计学意义(P<0.05)。结论 BALB/c小鼠经rSjLAP和rSjFBPA辅以福氏佐剂或纳米C60佐剂免疫,获得了一定的免疫保护力;福氏佐剂和纳米C60佐剂均增强了重组疫苗的免疫效果,但两种佐剂的免疫增强效果差异无统计学意义。

日本血吸虫Sj-Ts4样蛋白基因的克隆、表达和初步鉴定

目的为了寻找血吸虫病新的免疫学诊断候选分子,在大肠杆菌中高效表达日本血吸虫Sj-Ts4样融合蛋白。方法提取日本血吸虫成虫RNA,设计引物,用RT-PCR法扩增出日本血吸虫Sj-Ts4样蛋白基因编码序列,T载体连接,将其亚克隆入PGEX-5X-1载体中,经酶切、测序证实正确后,转化入大肠杆菌DH5α并用IPTG对该重组菌诱导表达,SDS-PAGE和Western blot方法观察表达结果。结果RT-PCR法扩增出一条大小约为870bp的片段,表达质粒PGEX-5X-1-SjTs4经双酶切和以该重组质粒为模板进行PCR扩增,均可获得一条与PCR产物一致的DNA片段,诱导表达后,经SDS-PAGE可见一条约58ku大小的GST融合蛋白条带,并可与抗GST特异性抗体反应。结论本实验成功地克隆了日本血吸虫Sj-Ts4样抗原的编码基因,并在原核细胞中进行表达,为进一步验证其免疫诊断价值奠定了基础。

抗rSj14-3-3单克隆抗体检测循环抗原及疗效考核价值的初步研究

目的 探讨抗重组日本血吸虫 14 3 3(rSj14 3 3)的单克隆抗体检测血吸虫循环抗原对血吸虫病疗效考核的价值。方法 制备纯化的抗日本血吸虫 14 3 3的单克隆抗体 ,以纯化的兔抗rSj14 3 3多抗和酶标羊抗兔多抗为检测系统的酶联免疫吸附试验 (P M ELISA)法 ,分别对同批治疗前及治疗后 2、4、6、12个月的血吸虫病患者和感染兔血清进行检测 ,并与常规检测抗体的可溶性虫卵抗原 -酶联免疫吸附试验 (SEA ELISA)法比较。结果 P M ELISA法对治疗前血吸虫病患者检测阳性率为 98 2 % ,而检测抗体的SEA ELISA为 95 5 %。P M ELISA法对治愈后 2、4、6、12个月血清循环抗原检测阴转率分别为 17 7%、4 1 9%、5 5 %、85 % ,而检测抗体的SEA ELISA法分别为 3 2 %、6 5 %、12 5 %、15 %。结论 该法既有较好的诊断价值 ,又有较好的疗效考核价值。

Selection of Immunogenic Peptide Mimics of Male Worm Origin of Schistosoma Japonicum using Phage Display Peptide Library

To select the immunogenic short peptide mimics of male worm origin of Schistosoma japonicum (Sj) and to explore their protection effect against schistosomiasis in mice, the random phage display peptide library of 12-mer was screened with IgG to soluble male worm antigen of Sj, and the specific positive clones selected through three rounds of screenings were detected by Dot-ELISA, and then injected subcutaneously into mice for vaccination and protection assessment against Sj. It was found that 18 randomly picked phage displayed clones all showed definite antigenicity with various intensities. The pooled phages displayed clones could induce production of specific antibodies and cause 31.72% of worm reduction rate and 51.54% of egg reduction rate in mice, revealing a significant difference (P<0.001) in comparison with those of the controls. It concludes that the short peptide mimics of male worm origin of Sj obtained by affinity screening phage display peptide library can elicit partial protection against this pathogen.