Coexistence of sense and anti-sense mRNAs of variant surface protein in Giardia lamblia trophozoites
Coexistence of sense and anti-sense mRNAs of variant surface protein in Giardia lamblia trophozoites
复制标题
贾第鞭毛虫滋养体中变异表面蛋白的正义和反义 mRNA 的共存
DOI：
10.1016/j.bbrc.2014.01.074
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发表时间：
2014-02-14
影响因子：
3.1
通讯作者：
Feng Xianmin
中科院分区：
文献类型：
--
作者：
Guo Junli;Zheng Wenyu;Feng Xianmin
蓝氏贾第鞭毛虫（Giardia lamblia）是一种寄生性原生动物，其表面抗原是变异表面蛋白（VSP）家族的成员，其逃避宿主免疫系统攻击的策略是周期性变化。一个转录后基因沉默机制已被提出来解释在任何时候只有一个在许多可能的VSP的存在。为了进一步研究这一现象，我们从培养的滋养体中提取总RNA。Lamblia C2分离物，并从RNA逆转录cDNA。利用巢式PCR技术从总cDNA中扩增正义和反义VSPs，引物从cDNA文库的3 '保守区和已知的5'或3'末端设计。扩增产物的序列分析显示超过34个全长反义VSP和一个有义VSP的涂片。序列比对和比较表明，这些VSPs含有可变的N-末端和保守的C-末端，根据N-末端序列的大小和变异可分为5个进化枝。所有反义VSP均以有义形式存在，但无相应的反义VSP存在于有义RNA（snsRNA）16中。培养的G. Lamblia支持VSP表达的转录后调节。我们提出，VSP同时以正义和反义形式转录形成双链RNA（dsRNA），其被Dicer内切核酸酶降解，而没有反义转录的VSP（例如，snsRNA 16）将在贾第虫的表面上表达。此外，在调查过程中，还发现了以前不知道的VSP。PCR扩增特异性正义和反义VSP cDNA可用于鉴定G.这比使用特异性单克隆抗体方法更容易。(C)由Elsevier Inc.出版。
A strategy of the parasitic protozoan Giardia lamblia to evade attack from the host immune system is periodic changes of its surface antigen, a member of the variant surface protein (VSP) family. A post-transcriptional gene silencing mechanism has been proposed to explain the presence of only one among many possible VSPs at any time. To investigate this phenomenon further, we extracted total RNA from cultured trophozoites of the G. lamblia C2 isolate, and cDNA was reverse-transcribed from the RNA. Sense and anti-sense VSPs were amplified from the total cDNA using nested PCR with primers designed from the 3'-conserved region and the known 5' or 3' end of the cDNA library. Sequence analyses of the amplified products revealed more than 34 full-length antisense VSPs and a smear of sense VSPs. Sequence alignments and comparisons revealed that these VSPs contained variable N-termini and conserved C-termini, and could be classified into 5 clades based on the sizes and variations of the N-terminal sequence. All antisense VSPs existed in the sense forms, but no corresponding antisense VSP existed for sense RNA (snsRNA) 16. The coexistence of sense and antisense VSP mRNAs in cultured G. lamblia supports the post-transcriptional regulation of VSP expression. We propose that VSPs transcribed simultaneously in the sense and antisense forms form double-stranded RNAs (dsRNAs) which are degraded by the Dicer endonuclease, while a VSP without an antisense transcription (e.g., snsRNA16) will be expressed on the surface of Giardia. In addition, in the course of this investigation VSPs were identified that were previously not known. PCR-based amplification of specific sense and antisense VSP cDNAs can be used to identify the specific VSP on G. lamblia trophozoites, which is easier than using specific monoclonal antibody approaches. (C) 2014 Published by Elsevier Inc.