RHD基因不同转录子表达载体构建

发布时间：2018-03-17 02:29

本文选题：RHD基因　切入点：转录子　出处：《大连医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：目前，Rh(D)抗原在红细胞膜的表达机制仍不清楚，研究发现其表达可能依赖于一个复合体。其中，RHD基因存在多种不同的另路剪接转录子，它们很可能参与了D抗原的表达。本实验分别构建RHD基因几种不同转录子的真核重组表达载体，其中包括正常RHDcDNA，以及和正常RhD mRNA序列最相近的DEL9、DEL89转录子，期望为探讨Rh(D)抗原表达机制及复合体组成成分奠定相关基础。方法：收集Rh(D)阳性新生儿脐血，利用血清学及分子生物学方法分别鉴定其Rh表型和基因型：盐水试管法鉴定其Rh表型，包括Rh D、C、c、E和e抗原；RHD基因型的鉴定则通过提取样本DNA，用检测部分D型的试剂盒对RHD基因10个外显子进行检测，以确认其实属于包含所有10个外显子的Rh(D)阳性样本。利用全血总RNA提取试剂盒从此脐血网织红细胞中提取总RNA，根据设计的不同引物，分别采用一步法和两步法进行RT-PCR扩增，通过琼脂糖凝胶电泳分离目的基因片段，再切胶并溶胶纯化目的基因；将纯化后的基因片段连接至TOPO TA克隆测序载体pCR4-TOPO，通过转化至TOP10大肠杆菌、以适量菌液均匀涂布至含氨苄青霉素的普通琼脂平板，37℃倒置过夜培养，，其后，随机挑取单一菌落至含氨苄青霉素的液体培养基中37℃水平震摇过夜增菌筛选培养，随后以碱裂解法提取纯化质粒，再对提取的质粒进行测序，挑选出序列完整无突变的质粒；以该筛选质粒为模板，使用设计的新引物进行PCR，扩增为构建表达载体用的目的基因，经琼脂糖凝胶电泳后切胶纯化，再将其连接至真核表达载体pcDNA3.1/V5-His-TOPO，通过再次转化至大肠杆菌、筛选增菌培养，提取并纯化质粒，最后经质粒测序筛选出含有序列正确、无突变目的基因，且插入方向正确的重组质粒。结果：血清学盐水试管法初步检测结果显示2份样本均为Rh(D)阳性，Rh表型分别为DCcEe和DCCee；基因分型结果再次验证两样本均为Rh(D)阳性样本：没有发生外显子缺失。其后，成功提取样本总RNA，一步法RT-PCR扩增共获得五个转录子条带，分别为正常RHDcDNA，DEL9，DEL89，DEL79及DEL789。由于目的条带较弱，随后采用两步法RT-PCR获得正常RHDcDNA条带，紫外灯下切胶后溶胶纯化，而DEL9和DEL89转录子则直接合成。含上下游非编码区的RHDcDNA成功克隆至TOPO TA克隆测序载体，经过克隆筛选、质粒序列分析结果显示：获得的片段中含有正常无突变RHD基因编码区序列（包含基因上下游非编码区的序列），以此含RHDcDNA的筛选质粒为模板，经PCR扩增获得从起始密码子至终止密码子的RHD基因编码区全长序列（不含上下游非编码区）。之后将大量扩增后获得的RHDcDNA、以及DEL9和DEL89分别成功克隆至pcDNA3.1/V5-His-TOPO真核表达载体，经过筛选克隆、测序验证，三种目的基因片段序列完整无突变且插入方向均正确，从而成功构建3种不同RHD转录子的真核表达载体。结论：实验成功构建了正常RHDcDNA、以及DEL9和DEL89转录子的真核表达载体。RHD基因不同转录子表达载体的构建为进一步研究Rh(D)抗原的膜表达机理奠定了基础。DEL9及DEL89转录子的表达载体构建为进一步研究D放散型（Del）个体红细胞Rh(D)膜抗原表达机理奠定了基础。
[Abstract]:Objective: at present, Rh (D) expression mechanism of antigens in the red cell membrane is still unclear, the research found its expression may depend on a complex. Among them, there are several different RHD gene alternative splicing transcripts, their expression might be involved in D antigen. The eukaryotic recombinant RHD gene were constructed in this experiment several different transcript expression vectors, including normal RHDcDNA and normal RhD and mRNA sequences most similar DEL9, DEL89 transcripts, expectations for the study of Rh (D) antigen expression mechanism and complex composition to lay the foundation.
Methods: Rh (D) positive neonatal umbilical cord blood, respectively, and identified the Rh phenotype and genotype by serological and molecular biological methods: saline tube method to identify their Rh phenotypes, including Rh D, C, C, E and e antigen; identification of RHD genotypes by extracting samples DNA, D type kit detection of 10 exons of RHD gene were detected to confirm are actually contains all 10 exons of the Rh (D) positive samples. The whole blood total RNA extraction kit from umbilical cord blood reticulocyte extract total RNA in red blood cells, according to the different primers were designed, respectively, by one step and two steps for RT-PCR amplification, separation of target gene fragment by agarose gel electrophoresis, gel sol and cut purified gene; gene fragment of purified TOPO is connected to TA through cloning and sequencing vector pCR4-TOPO and transformed into TOP10, with the proper amount of bacteria liquid evenly to contain The ordinary agar ampicillin, 37 C inverted overnight culture, then randomly selected single colony to ampicillin containing liquid culture medium overnight jolt 37 C level increased screening of bacteria culture, followed by alkaline lysis and purification of plasmid, plasmid was extracted, sequenced, selected plasmid sequence complete mutation; in the screening of new plasmid as template, using primers designed by PCR amplification of target gene expression vector used for the construction, after agarose gel electrophoresis purification, and then connect it to the eukaryotic expression vector pcDNA3.1/ through V5-His-TOPO, once again transformed into E.coli, screening and culturing, extraction and purification of plasmid, finally the plasmid sequencing screened contains correct sequence mutation gene, and the recombinant plasmid was inserted into the right direction.
Results: preliminary results of serological detection assay showed that the saline tube 2 samples were Rh positive (D), Rh and DCcEe phenotypes were DCCee; genotyping results of two samples were tested again Rh (D) positive samples: no exon deletion. Subsequently, the successful extraction of total RNA samples, one step RT-PCR a total of five transcripts amplified bands, respectively RHDcDNA, DEL9, DEL89, DEL79 and DEL789. due to the weak band, followed by the two step RT-PCR normal RHDcDNA bands under UV light after sol gel purification, while DEL9 and DEL89 transcripts were directly synthesized containing downstream non encoding. The RHDcDNA was successfully cloned to the TOPO vector by TA clone sequencing, clone screening, plasmid sequence analysis showed that the obtained fragment containing normal no mutations in the RHD gene encoding region sequence (the sequence contains genes downstream non encoding regions), which contains RHDcDNA The screening of plasmid as template, amplified by PCR from the start codon to the full-length encoding region of RHD gene stop codon (excluding the downstream non encoding region). After a large number of amplified from RHDcDNA, and DEL9 and DEL89 were successfully cloned into eukaryotic expression vector pcDNA3.1/V5-His-TOPO. After screening and cloning, sequencing three, the target gene fragment sequence mutation and insertion direction are correct, the eukaryotic expression vector and 3 different RHD transcripts was successfully constructed.
Conclusion: the successful construction of the normal RHDcDNA, and the DEL9 and DEL89 transcription of eukaryotic expression vector of.RHD gene transcripts in different expression vectors for the further study of Rh (D) antigen expression mechanism of membrane expression vector laid the foundation of.DEL9 and DEL89 transcripts of the construction for the further research of D subtype (Del) individual Rh of red blood cell (D) membrane antigen expression mechanism laid the foundation.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R3416

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