[发明专利]日本血吸虫（中国大陆株）MFS4基因的克隆、表达和DNA疫苗

摘要

本发明涉及生物基因领域，具体涉及日本血吸虫(中国大陆株)MFS4基因的基因序列、免疫筛选、在大肠杆菌中的表达、DNA疫苗的构建及小鼠免疫保护试验。本发明基于我们对东方田鼠抗日本血吸虫病机理研究的发现，用健康东方田鼠血清IgG3抗体免疫筛选日本血吸虫成虫cDNA文库，获得一个阳性克隆，用RACE技术扩增出含ORF的基因。同源性序列分析表明其为一个新基因(MFS4)，开放阅读框含780bp，编码259个氨基酸。将MFS4基因亚克隆至原核表达质粒pET28a(+)中，构建重组表达质粒pET28a/MFS4，在BL21(DE3)表达菌中获得高效表达，融合蛋白分子量约35kD，具有良好的抗原性；将该基因亚克隆至真核表达质粒pcDNA3中，构建成重组质粒pcDNA3/MFS4，制备DNA疫苗免疫昆明系小鼠，获得28.6％的减虫率、21.73％的减卵率。

主权项

1.一种编码日本血吸虫(中国大陆株)MFS4基因的核苷酸序列，其特征在于它具有如下的核苷酸序列及相应的氨基酸序列：gtatcatatc gttaacgcgc tccagttttg tttttgccca ggtattata atg gcg gtt 58 Met Ala Val 1ggt ggt cag aaa aag gtc act aaa ggg ggg aag aaa gga ggc aaa aag 106Gly Gly Gln Lys Lys Val Thr Lys Gly Gly Lys Lys Gly Gly Lys Lys5 10 15aaa acg gct gat cca ttc tct aaa aag gag tgg tat gat atc aag gct 154Lys Thr Ala Asp Pro Phe Ser Lys Lys Glu Trp Tyr Asp Ile Lys Ala20 25 30 35cca gct atg ttt cct aaa agg aca tgt gca aga acg ctt gtc aca cga 202Pro Ala Met Phe Pro Lys Arg Thr Cys Ala Arg Thr Leu Val Thr Arg 40 45 50act cag ggt act cgc ata gcc agt gaa gca ctt aag ggt cgt gtt gtt 250Thr Gln Gly Thr Arg Ile Ala Ser Glu Ala Leu Lys Gly Arg Val Val 55 60 65aca ctt agt ctt ggg gat ctg agc gaa aag aac gaa gaa gtc ttc cgt 298Thr Leu Ser Leu Gly Asp Leu Ser Glu Lys Asn Glu Glu Val Phe Arg 70 75 80aaa ttc aag ctt caa atc gaa gat gtc cag ggg cgt cat tgt cta aca 346Lys Phe Lys Leu Gln Ile Glu Asp Val Gln Gly Arg His Cys Leu Thr85 90 95aat ttc cac gga tta gaa ctt act cga gac aaa cta tgt tca gtt gtt 394Asn Phe His Gly Leu Glu Leu Thr Arg Asp Lys Leu Cys Ser Val Val100 105 110 115gtt aag tgg cgg tca act ata gaa gcg cac gtc gac gtc aaa aca aca 442Val Lys Trp Arg Ser Thr Ile Glu Ala His Val Asp Val Lys Thr Thr 120 125 130gat gga tac tta ctt cgt ttc ttt att atc acg ttc act cca agt gta 490Asp Gly Tyr Leu Leu Arg Phe Phe Ile Ile Thr Phe Thr Pro Ser Val 135 140 145cca cgg tct gaa agg ctc cac tct tat gct cag acc aca cgc ata aaa 538Pro Arg Ser Glu Arg Leu His Ser Tyr Ala Gln Thr Thr Arg Ile Lys 150 155 160cgg att cgc gct cgc ctt gtt gag ata atc cag caa gag gtt tct act 586Arg Ile Arg Ala Arg Leu Val Glu Ile Ile Gln Gln Glu Val Ser Thr165 170 175tgc gat ctg aaa gag gtc gtt aat aag ctt att cca gat tcc att gca 634Cys Asp Leu Lys Glu Val Val Asn Lys Leu Ile Pro Asp Ser Ile Ala180 185 190 195caa gac gct cga aaa gcg gca tca tgg ata tat cca cta ggt gag aca 682Gln Asp Ala Arg Lys Ala Ala Ser Trp Ile Tyr Pro Leu Gly Glu Thr 200 205 210ttt atc cgc aaa gtt aag gtt tta aaa aga cca aaa gct gat ctc gct 730Phe Ile Arg Lys Val Lys Val Leu Lys Arg Pro Lys Ala Asp Leu Ala 215 220 225cgg ctc atg gaa ctt cat ggt gaa tca aag tca act gcc cct ggt gag 778Arg Leu Met Glu Leu His Gly Glu Ser Lys Ser Thr Ala Pro Gly Glu 230 235 240act gta tcc cgt cca gat cac tat gaa cct ccg aaa gtt gat tca gtg 826Thr Val Ser Arg Pro Asp His Tyr Glu Pro Pro Lys Val Asp Ser Val245 250 255taattcaata aagaatttca tccggaaaaa aaaaaaaaaa aaaaa 871