简化的差示PCR技术在伯氏疟原虫氯喹抗性基因分离中的应用

APPLICATION OF SIMPLIFIED DIFFERENTIAL DISPLAY POLYMERASE CHAIN REACTIONTO ISOLATE CHLOROQUINE-RESISTANT RELATED GENES OFPLASMODIUM BERGHEI

目的 为探察伯氏疟原虫氯喹抗性株 (RC株 )和敏感株 (N株 )的遗传学差异。方法 先维持对 RC株连续大剂量氯喹治疗 (5 0 m g/ (kg.d) ,再通过简化逆转录差示多聚酶链反应技术 (s DDRT- PCR)寻找 RC株和 N株疟原虫 c DNA差异片段并对所产生的可疑差异片段进行反复的 DNA回收和 PCR鉴定 ,然后选其肯定的差异片段 (N2 5 和 R2 5 )作克隆测序和序列分析及同源性检索。结果 尽管差异片段 N2 5 和 R2 5 的克隆分子 N2 5 2 和 R2 5 1 的 DNA序列相同性为 99.8% ,但由于多点突变导致它们的编码氨基酸及其模拟二级结构出现很大差异。BL AST(2 .0 6 )检索未能在基因库中发现与 R2 5 1 和 N2 5 2 相似的基因序列 ,不过它们的部分核苷酸序列 (从 12 8nt到 189nt)与褐鼠磷脂酶 Bm RNA部分序列 (从 10 5 3nt到 1114nt)具有高度同源性 :在 R2 5 1 和N2 5 2 分别为 93.5 %和 91.9%。结论 简化的差示 PCR技术结合差异片段的反复验证和序列分析可用于氯喹抗性相关基因的分离。

Aim To insight into genetic differences between chloroquine-resistant line (RC) and chloroquine-sensitive strain(N) of Plasmodium berghei.Methods\ After continous chloroquine-pressure upon RC line at higher dosage(50mg/kg·d),total RNAs from the RC line and the N strain of P.berghei were isolated for simplified differential display reverse transcript polymerase chain reaction(sDDRT-PCR).The generated differential fragments had been repetitively rescued and identified by PCRs before one pair of suspected differential fragments(N 25 and R 25 )were cloned and sequenced.Then,their sequences were analyzed through PC-gene program and BLAST search.Results Though the identity of two nucleotide sequences of N 252 and R 251,cloned separately from the N 25 and R 25 fragments, were up to 99.8%,their predicted amino acid secondary structures were quite different due to multiple mutations.Compared with the published sequences in GeneBank,EMBL,DDBJ and PDB database,no similar gene was found,using BLAST search.However their partial nucleotide sequences(62nt from query 128nt to189nt bore highly homology to part sequence(from 1053nt to1114nt)of rattus norvegicus mRNA for phospholipase B,up to 93.5% in N 251 and 91.9% in N 252 respectively.Conclusion It is feasible to isolate chloroquine-resistant related genes by using simplified DDRT-PCR combined repetitively rescuing and PCR identifying the interest differential DNAs together with sequence analyses.