人卵巢癌cDNA噬菌体表达文库的构建及鉴定

Construction and identification of human ovarian carcinoma cDNA phage expression library

目的构建人卵巢癌cDNA噬菌体表达文库。方法培养人卵巢肉瘤样癌细胞系细胞,提取总RNA,在RNA转录本5′末端开关机制(switching mechanismat5′end of RNAtranscript,SMART)寡核苷酸参与下,反转录合成cDNA第一链,用长距离聚合酶链式反应(long-distance PCR,LD-PCR)合成cDNA第二链并扩增双链DNA,鉴定扩增产物。采用层析离心柱除去小于500bp的片段后,与去磷酸化的SfiⅠ酶消化的λTriplEx2噬菌体载体连接,用GigapackⅢGold包装提取物体外包装噬菌体,感染宿主菌XL1-Blue,滴定原始文库以确定库容量大小,用蓝白斑试验测定重组率。随机挑取12个白色噬斑,采用平板裂解法扩增,用λ噬菌体DNA纯化试剂盒提取、纯化DNA,用SfiⅠ酶切鉴定插入片段大小。扩增原始库后保存备用。结果 PCR产物琼脂糖凝胶电泳在400bp^7kb的弥散背景下,可见到数条对应于优势转录本的特征性条带,符合哺乳动物细胞的特征。构建成含5×106pfu/ml的人卵巢癌cDNA噬菌体表达文库,重组率为99.0%,插入外源cDNA片段大小范围500bp^4kb,平均长度约1.79kb。扩增库滴度2.7×109pfu/ml。结论成功构建人卵巢癌cDNA噬菌体表达文库,可进一步用于筛选卵巢癌相关抗原基因。

Objective To construct a human ovarian carcinoma cDNA phage expression library for screening tumor antigens. Methods The human ovarian sarcomatiod carcinoma cell line BUPH ： OVSC- 1 was cultured and total RNA was isolated. In the presence of switching mechanism at 5＇ end of RNA transcript oligonucleotide, the single- strand and double - strand of cDNA were synthesized through reverse transcription and long distance PCR. After removed the fragments smaller than 500 bp, the remaining cDNAs were combined with the dephosphorylated γTriplEx2 phage vector. The ligated cDNAs were packaged into phage particles in vitro with Gigapack III gold pack aging extract. A small portion of the packaged phage was used to infected E. coli XL1 - Blue and the primary library was titrated and the percentage of recombinant clones was determined by blue white test. Sfi I restriction endonucleases was used to identify the size of inserted cDNA. The primary library was amplified for better preserving. Results Agarose gel electrophoresis of PCR product showed several characteristic bands corresponds to advantage transcript in the background of 400 bp-7 kb smear, which according to mammals cell feature. The primary library consisted of 5.0 × 10 6 pfu/ml and recombinant rate was 99.0%. The length range of the inserts was between 500 bp - 4 kb with the average of 1.79 kb. The amplified library titration was 2.7 × 10 9 pfu/ml. Conclusions A high quality human ovarian carcinoma cDNA phage expression library derived from ovarian sarcomatiod carcinoma cell line is constructed, which may be used to screen the ovarian tumor-associated antigens.