东方田鼠肝、肺脏噬菌体展示cDNA文库的构建、筛选及克隆分析
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摘要
日本血吸虫病是我国一种危害严重的人畜共患寄生虫病。东方田鼠是迄今在疫区发现的唯一一种感染血吸虫后不致病的哺乳类动物,但目前对其抗病的分子机制仍不清楚。噬菌体展示技术是近年建立和发展起来的利用噬菌体表达外源基因的一项新技术。本研究通过构建东方田鼠肝脏、肺脏T7噬菌体展示cDNA文库,并用日本血吸虫童虫可溶性裂解产物进行筛选,以寻找东方田鼠抗日本血吸虫病相关因子,为揭示其分子机理奠定基础,开拓思路。
1.构建东方田鼠肝脏、肺脏T7噬菌体展示cDNA文库。
1.1用Trizol试剂提取东方田鼠肝脏、肺脏总RNA,分离纯化mRNA,经反转录合成双链cDNA。在双链cDNA末端加上EcoRⅠ/HindⅢ定向接头并用EcoRⅠ和HindⅢ酶切,使其两端分别带EcoRⅠ和HindⅢ粘性末端。用Mini Column纯化,收集300bp以上的双链cDNA片段,再连接于带有EcoRⅠ和HindⅢ末端的T7 Select 10 3b载体,经体外包装后,以BLT5403为受体菌构建东方田鼠肝脏、肺脏T7噬菌体展示cDNA文库。
1.2经测定,肝文库库容量为1.3×10~7 pfu/mL,扩增后文库滴度为1.8×10~(11)pfu/mL。对从原始文库中随机挑取的100个噬菌斑进行PCR鉴定,重组率为91.7%,阳性克隆片段大小分布在200-1000bp,其中有95.5%的插入片段大于300bp。肺文库库容量为1.5×10~6pfu/mL,扩增后文库滴度为1.1×10~(12)pfu/mL。对从原始文库中随机挑取的100个噬菌斑进行PCR鉴定,重组率为91%,阳性克隆片段大小分布在200-1500bp,其中有90%的插入片段大于300bp。
2.日本血吸虫童虫裂解物对东方田鼠肝脏T7噬菌体展示cDNA文库的筛选及克隆分析
2.1以日本血吸虫童虫可溶性裂解物为探针,筛选东方田鼠肝脏噬菌体展示cDNA文库,对部分阳性克隆的插入片段进行PCR鉴定及测序。通过互联网对测序获得的核苷酸序列进行同源性分析,并预测新基因编码蛋白的结构与功能。将阳性克隆与童虫一起培养,观察体外杀伤情况。
2.2筛选获19个有效阳性克隆,其中有13个EST序列与已知基因或表达序列标签同源,其中包括5种蛋白酶:氢哌啶羧酸氧化酶、细胞色素C氧化酶、alpha-2-HS-glycoprotein、脂酰辅酶A脱氢酶、DNA拓扑异构酶II;5种功能结合蛋白:维生素D结合蛋白、具有R3H结构域的一种mRNA结合蛋白、载脂蛋白E、多聚胞嘧啶结合蛋白、M4蛋白;2种信号转导蛋白:整合素alpha 8、CASP8和FADD类似性细胞程序性死亡调节蛋白;1种细胞组成蛋白:核糖体蛋白S18。6个EST序列与已知基因或表达序列标签均无同源性,为新的表达序列标签。所得阳性克隆插入cDNA片段大小分布在300～800bp之间。将这些阳性噬菌体克隆和血吸虫童虫共培养,所有克隆对童虫的杀伤效果是空白对照的1.54到2.60倍,为空载体噬菌体对照的1.06至1.79倍。
本实验成功构建了东方田鼠肝、肺脏T7噬菌体展示cDNA文库。筛选东方田鼠肝脏T7噬菌体展示cDNA文库所获得的阳性克隆与童虫共培养产生了不同程度杀伤童虫作用,提示筛选到的某些蛋白可能与东方田鼠抗日本血吸虫病相关,对相关基因的功能作进一步地深入研究,将为寻找东方田鼠抗日本血吸虫病相关分子,查明东方田鼠抗病机制提供重要基础。
Schistosoma japonicum is agent of zoonotic infection which can result in serious disease both humans being and in the domestic animal reservoir hosts. Schistosomiasis remains a major public health problem in China. Microtus fortis is the only one mammalian animals with resistance to Schistosomiasis found in the epidemic area in China. Here, we reported the Construction of two T7 phage display cDNA libraries from liver and lung of Microtus fortis. The liver library was screened to find the schistosomiasis-resistence-related gene of Microtus fortis.
1 Construction of two T7 phage display cDNA libraries from liver and lung of Microtus fortis.
1.1 The mRNA was isolated from total RNA from livers and lung of Microtus fortis by Trizol reagent, and used to synthesize the ds cDNA by reverse transcription. Then the ds cDNA was given EcoRⅠand HindⅢadhering ends by ligation with the directional EcoRⅠ/ HindⅢlinkers and digestion with EcoRⅠand HindⅢ. The ds cDNA fragments longer than 300bp in length were fractionated by Mini Column, and ligated into the T7 Select 10-3b vertor with EcoRⅠand HindⅢadhering ends. After packaging in vitro, the recombinant T7 Select 10-3b was transformed into BLT5403 to construct a T7 phage display cDNA library.
1.2 The liver library constructed here contained 1.3×10~7 clones and the titer of the amplied library was 1.8×10~(11) pfu/ mL. The PCR identification results of 100 clones picked at random showed that 91.7% clones were recombinant and 95.5 % of recombinant clones contained cDNA fragments longer than 300bp in length. The lung library constructed here contained 1.5×10~6 clones and the titer of the amplied library was 1.1×10~(12) pfu/ mL. The PCR identification results of 100 clones picked at random showed that 91% clones were recombinant and 90 % of recombinant clones contained cDNA fragments longer than 300bp in length.
2 The screening of T7 phage display cDNA library from liver of Microtus fortis with schistosomulua.
2.1 The T7 phage display cDNA library from liver of Microtus fortis was screened with the soluble lysate of schistosomulua. Positive clones were identified firstly by PCR and further by sequencing and data analysis through internet BLASTX software of NCBI and Expert Protein Analysis System of expasy and interproscan.
2.2 19 available positive clones were obtained and their PCR product sizes ranged from 200～800bp. 13 ESTs, which contain 5 enzymatic activity proteins、5 binding proteins、2 signal transducers and 1 structural constituent protein, were found in NCBI, while 6 ESTs were not found in GenBank. When co-cultured with schistosomulua, the mortality of schistosomulua induced by all of the available positive clones were 1.54-2.60 times than blank control, and 1.06-1.79 times than negative phage control.
The results suggested that the T7 phage display cDNA libraries from liver and lung of Microtus fortis were successfully constructed. To Screen The T7 phage display cDNA library from liver of Microtus fortis with schistosomulua,Some of gained clones maight be the schistosomiasis-resistence-related gene of Microtus fortis.
1.构建东方田鼠肝脏、肺脏T7噬菌体展示cDNA文库。
1.1用Trizol试剂提取东方田鼠肝脏、肺脏总RNA,分离纯化mRNA,经反转录合成双链cDNA。在双链cDNA末端加上EcoRⅠ/HindⅢ定向接头并用EcoRⅠ和HindⅢ酶切,使其两端分别带EcoRⅠ和HindⅢ粘性末端。用Mini Column纯化,收集300bp以上的双链cDNA片段,再连接于带有EcoRⅠ和HindⅢ末端的T7 Select 10 3b载体,经体外包装后,以BLT5403为受体菌构建东方田鼠肝脏、肺脏T7噬菌体展示cDNA文库。
1.2经测定,肝文库库容量为1.3×10~7 pfu/mL,扩增后文库滴度为1.8×10~(11)pfu/mL。对从原始文库中随机挑取的100个噬菌斑进行PCR鉴定,重组率为91.7%,阳性克隆片段大小分布在200-1000bp,其中有95.5%的插入片段大于300bp。肺文库库容量为1.5×10~6pfu/mL,扩增后文库滴度为1.1×10~(12)pfu/mL。对从原始文库中随机挑取的100个噬菌斑进行PCR鉴定,重组率为91%,阳性克隆片段大小分布在200-1500bp,其中有90%的插入片段大于300bp。
2.日本血吸虫童虫裂解物对东方田鼠肝脏T7噬菌体展示cDNA文库的筛选及克隆分析
2.1以日本血吸虫童虫可溶性裂解物为探针,筛选东方田鼠肝脏噬菌体展示cDNA文库,对部分阳性克隆的插入片段进行PCR鉴定及测序。通过互联网对测序获得的核苷酸序列进行同源性分析,并预测新基因编码蛋白的结构与功能。将阳性克隆与童虫一起培养,观察体外杀伤情况。
2.2筛选获19个有效阳性克隆,其中有13个EST序列与已知基因或表达序列标签同源,其中包括5种蛋白酶:氢哌啶羧酸氧化酶、细胞色素C氧化酶、alpha-2-HS-glycoprotein、脂酰辅酶A脱氢酶、DNA拓扑异构酶II;5种功能结合蛋白:维生素D结合蛋白、具有R3H结构域的一种mRNA结合蛋白、载脂蛋白E、多聚胞嘧啶结合蛋白、M4蛋白;2种信号转导蛋白:整合素alpha 8、CASP8和FADD类似性细胞程序性死亡调节蛋白;1种细胞组成蛋白:核糖体蛋白S18。6个EST序列与已知基因或表达序列标签均无同源性,为新的表达序列标签。所得阳性克隆插入cDNA片段大小分布在300～800bp之间。将这些阳性噬菌体克隆和血吸虫童虫共培养,所有克隆对童虫的杀伤效果是空白对照的1.54到2.60倍,为空载体噬菌体对照的1.06至1.79倍。
本实验成功构建了东方田鼠肝、肺脏T7噬菌体展示cDNA文库。筛选东方田鼠肝脏T7噬菌体展示cDNA文库所获得的阳性克隆与童虫共培养产生了不同程度杀伤童虫作用,提示筛选到的某些蛋白可能与东方田鼠抗日本血吸虫病相关,对相关基因的功能作进一步地深入研究,将为寻找东方田鼠抗日本血吸虫病相关分子,查明东方田鼠抗病机制提供重要基础。
Schistosoma japonicum is agent of zoonotic infection which can result in serious disease both humans being and in the domestic animal reservoir hosts. Schistosomiasis remains a major public health problem in China. Microtus fortis is the only one mammalian animals with resistance to Schistosomiasis found in the epidemic area in China. Here, we reported the Construction of two T7 phage display cDNA libraries from liver and lung of Microtus fortis. The liver library was screened to find the schistosomiasis-resistence-related gene of Microtus fortis.
1 Construction of two T7 phage display cDNA libraries from liver and lung of Microtus fortis.
1.1 The mRNA was isolated from total RNA from livers and lung of Microtus fortis by Trizol reagent, and used to synthesize the ds cDNA by reverse transcription. Then the ds cDNA was given EcoRⅠand HindⅢadhering ends by ligation with the directional EcoRⅠ/ HindⅢlinkers and digestion with EcoRⅠand HindⅢ. The ds cDNA fragments longer than 300bp in length were fractionated by Mini Column, and ligated into the T7 Select 10-3b vertor with EcoRⅠand HindⅢadhering ends. After packaging in vitro, the recombinant T7 Select 10-3b was transformed into BLT5403 to construct a T7 phage display cDNA library.
1.2 The liver library constructed here contained 1.3×10~7 clones and the titer of the amplied library was 1.8×10~(11) pfu/ mL. The PCR identification results of 100 clones picked at random showed that 91.7% clones were recombinant and 95.5 % of recombinant clones contained cDNA fragments longer than 300bp in length. The lung library constructed here contained 1.5×10~6 clones and the titer of the amplied library was 1.1×10~(12) pfu/ mL. The PCR identification results of 100 clones picked at random showed that 91% clones were recombinant and 90 % of recombinant clones contained cDNA fragments longer than 300bp in length.
2 The screening of T7 phage display cDNA library from liver of Microtus fortis with schistosomulua.
2.1 The T7 phage display cDNA library from liver of Microtus fortis was screened with the soluble lysate of schistosomulua. Positive clones were identified firstly by PCR and further by sequencing and data analysis through internet BLASTX software of NCBI and Expert Protein Analysis System of expasy and interproscan.
2.2 19 available positive clones were obtained and their PCR product sizes ranged from 200～800bp. 13 ESTs, which contain 5 enzymatic activity proteins、5 binding proteins、2 signal transducers and 1 structural constituent protein, were found in NCBI, while 6 ESTs were not found in GenBank. When co-cultured with schistosomulua, the mortality of schistosomulua induced by all of the available positive clones were 1.54-2.60 times than blank control, and 1.06-1.79 times than negative phage control.
The results suggested that the T7 phage display cDNA libraries from liver and lung of Microtus fortis were successfully constructed. To Screen The T7 phage display cDNA library from liver of Microtus fortis with schistosomulua,Some of gained clones maight be the schistosomiasis-resistence-related gene of Microtus fortis.
引文
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