【作者】 江元山；

【导师】 胡维新；

【作者基本信息】 中南大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 L1在人类基因组中重复约50000次，占整个基因组DNA的约15％。在人类基因组中只有不到4000个全长L1。基因组全长L1长约6～7kb，该家族成员5＇末端往往为残端，L13＇末端多有长短不一的poly(A)尾。 哺乳动物全长L1包括含启动子的5＇端非翻译区，ORFⅠ和ORFⅡ两个非重叠蛋白编码区，3＇端非翻译区和poly(A)尾。ORFⅠ编码一个40kD的核酸结合蛋白；ORFⅡ编码一种具有核酸内切酶和逆转录酶活性的蛋白以及一个富半胱氨酸区。 本实验以ARH-77cDNA文库为模板PCR扩增出L1逆转录酶cDNA片段，并以此为探针，经菌落原位杂交得到L1逆转录酶DNA序列5＇端。随后，从ARH-77培养细胞中提取总RNA，进行RT-PCR获得L1逆转录酶DNA序列的3＇端及其poly(A)尾。 生物信息学分析菌落原位杂交筛选到的阳性克隆测序结果和RT-PCR测序结果，剪切、拼接出L1逆转录酶DNA序列。对本实验所得序列运用ORF finder进行阅读框预测并运用Conserved Domain Search分析表明该序列有一长度为552bp的开放性阅读框架，编码1条包含184个氨基酸残基的多肽链，其相对分子量约为21kD，为L1逆转录酶保守区氨基酸残基。 以此为依据设计引物扩增得到编码L1逆转录酶保守区的L1ORF DNA序列并把它与原核表达载体pQE30连接，得到重组原核表达质粒pQE30-L1ORF，转化到大肠杆菌JM109，经终浓度为1mmol/L的IPTG诱导表达，SDS-PAGE电泳检测发现在分子量21kD左右的位置有一随诱导时间延长而表达明显增强的蛋白泳带。 重组原核表达载体pQE30-L1ORF为融合蛋白表达载体，所表达的蛋白产物在氨基末端带有6个连续组氨酸标签，可以利用NI-NTA亲和层析柱简便地纯化，纯化后的L1融合蛋白产物经过透析处理后得到不含变性剂尿素的纯化L1融合蛋白产物用以作多克隆抗体制备试验。 初次免疫把弗氏完全佐剂与等体积的L1融合蛋白溶液研磨成油包水乳剂皮下多点注射于家兔的背部。随后的多次免疫和加强免疫是把弗氏不完全佐剂与等体积的L1融合蛋白溶液研磨成油包水乳剂皮下多点注射。经4～7次注射后，家兔将产生多克隆抗体，产生的硕士学位论文 中文摘要抗体用免疫扩散和 ELISA检测。更多还原

【Abstract】 Over 50 000 LI sequences exist in human genome, which accounts for as much as 15 % of human genome, 4 000 of these are full length, whereas the rest are truncated at the 5’ end. LI lacks long terminal repeats and is characterized by a poly(A) tail.A full-length mammalian LI sequence contains a 5’ untranslated region with a internal promotor, two nonoverlapping open reading frames (ORF I and ORFII ) ,and a 3’ untranslated region that ends in a poly(A) tail. ORF I encodes a RNA-binding protein, whereas ORF II encodes an endonuclease-reverse transcriptase activity and a cysteine rich domain.In this study, a 583 bp segment of L1 reverse transcriptase gene was amplified by PCR using human multiple myeloma cell line ARH-77 cDNA library as a template. 5’ end of the DNA sequence of L1 reverse transcriptase gene was obtained by in situ hybridization using L1 reverse transcriptase gene as a probe. Total RNA from the ARH-77 cell was isolated and obtained 3’ end and poly (A) of L1 reverse transcriptase gene by RT-PCR.We compared the sequences of #6 positive clones screened by in situ hybridization and the sequences obtained by RT-PCR, and analysed and spliced to obtain the DNA sequence of L1 reverse transcriptase gene. According to analysing by ORF finder and Conserved Domain Search in NCBI, the L1 reverse transcriptase gene in human multiple myeloma cell line ARH-77 has an open reading frame with 552 base pairs and encodes a 184 amino acid polypeptide with a theoretical 21 kD.A 552 bp sequence of L1 reverse transcriptase domain was acquired by PCR, then recombinant prokaryotic expression plasmid pQE30-L1 ORF was constructed by inserting LI ORF into prokaryotic expression vector pQE30. The expression of the recombinant plasmids was induced with 1 mmol/L IPTG. SDS-PAGE electrophores analysis showed that a new protein with molecular weight 21kD could be found. The expression level of L1 fusion protein was increased when IPTGconcentration was increased and would reach its highest expression at 6h.The prokaryotic expression vector pQE30 is fusion protein expression vector in which the target protein was expressed have been tagged with 6 consecutive histidine residues (6 xHis tag) in the N-terminal. The use of the affinity tags simplifies the purification of L1 fusion proteins by employing affinity chromatography methods.A rabbit was used to produce polyclonal antibodies of L1 fusion protein. In the first immune experiment, L1 fusion protein was blended with a same volume of Freund’s complete adjuvant and injected into the back of the rabbit by subcutaneous injection. In the rest of the immune experiments, L1 fusion protein was amalgamated with Freund’s incomplete adjuvant and injected into the back of the rabbit. After 4-7 times, polyclonal antibodies were produced and testified by immune diffusion and ELISA.更多还原