日本血吸虫新基因SjPP的筛选、克隆及对小鼠的免疫效果

Screening,Cloning and Immunological Efficiency of SjPP,a Novel Schistosoma japonicum Gene

目的筛选克隆东方田鼠血清识别的日本血吸虫靶抗原基因,并研究其免疫保护效果。方法利用东方田鼠感染血清免疫筛选日本血吸虫(中国大陆株)成虫cDNA表达文库获得阳性克隆,以5′RACE技术扩增获基因全长序列,构建含目的基因的核酸疫苗并研究在小鼠中的免疫保护效果。结果(1)获得4个日本血吸虫新的EST序列,即mfs-1(GenBank登录号BE974942)、mfs-3(GenBank登录号BE974944)、mfs-4(GenBank登录号BE974945)和mfs-5(GenBank登录号BE974946)。(2)mfs-5进行全长序列克隆,获得的日本血吸虫新基因SjPP(GenBank登录号AY902477)长1311bp,编码302个氨基酸,理论分子量为34.7kD,等电点为5.51;其氨基酸序列具有丝/苏氨酸蛋白磷酸酶(serine/threoninespecificproteinphosphatase,简称PP)的保守序列LRGNHE,并且含有该酶特异性抑制剂冈田酸的作用位点SAPNYC,与人的PP6、鼠PP6、果蝇PPⅤ等蛋白的氨基酸序列具有高度同源性,分别达72%、70%和70%。(3)成功构建核酸疫苗pCMV-Script/SjPP,免疫BALB/c小鼠诱导产生肝组织减卵率为23.91%,粪便减卵率为31.91%。结论(1)获得日本血吸虫新的抗原基因SjPP。(2)含SjPP基因的核酸疫苗在小鼠中诱导了部分免疫保护效果。

[Objective] The aim of the experiment is to screen and clone antigen genes of Schistosomajaponicum specific to the sera of Microtusfortis and study the protective efficiency. [ Method ] A cDNA library of Schistosomajaponicum adult worm was immunologically screened with the sera from Microtus fortis infected with the schistosome. Then mice were immuned with DNA vaccine of SjPP to evaluate the immunological protective efficiency. [Result]（1） Five positive clones, mfs-1, mfs-2, mfs-3, mfs-4 and mfs-5, were obtained and their sequences were deposited with GenBank under accession No. BE974942, BE974943, BE974944, BE974945, BE974946 respectively. （2） A full length cDNA fragment containing a complete open reading frame coding 302 amino acid sequences （Mr=34.7kD, pI=5.51） was amplified. Sequence analyses revealed that it was a novel gene which deduced amino acid sequence contain a conserve sequence （LRGNHE） of serine/threonine specific protein phosphatases, as well as a binding site （SAPNYC） of the phosphatase＇s specific inhibitor okadaic acid. The gene was showed high homology to other protein phosphatases such as human PP6 （72%）, PP4C （59%）, rat PPV （71%）, fruit fly PPV （70%）, fission yeast ppel （63%）, budding yeast sit4p （61%） and was named as SjPP （3） Compared with those of the blank control group, 23.91% reduction in mean hapetic tissue egg count and 31.91% reduction in mean faecal egg count were recovered in the mice injected with the recombinant plasmid pCMV-Script/SjPP. [ Conclusion ] （1） A novel antigen gene of Schistosomajaponicum, SjPP, was cloned and identified. （2） The partial protection was obtained in the mice vaccinated with the DNA vaccine pCMV-Script/SjPP.