本文選題：RHD基因　切入點(diǎn)：轉(zhuǎn)錄子　出處：《大連醫(yī)科大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：目前，Rh(D)抗原在紅細(xì)胞膜的表達(dá)機(jī)制仍不清楚，研究發(fā)現(xiàn)其表達(dá)可能依賴(lài)于一個(gè)復(fù)合體。其中，RHD基因存在多種不同的另路剪接轉(zhuǎn)錄子，它們很可能參與了D抗原的表達(dá)。本實(shí)驗(yàn)分別構(gòu)建RHD基因幾種不同轉(zhuǎn)錄子的真核重組表達(dá)載體，其中包括正常RHDcDNA，以及和正常RhD mRNA序列最相近的DEL9、DEL89轉(zhuǎn)錄子，期望為探討Rh(D)抗原表達(dá)機(jī)制及復(fù)合體組成成分奠定相關(guān)基礎(chǔ)。 方法：收集Rh(D)陽(yáng)性新生兒臍血，利用血清學(xué)及分子生物學(xué)方法分別鑒定其Rh表型和基因型：鹽水試管法鑒定其Rh表型，包括Rh D、C、c、E和e抗原；RHD基因型的鑒定則通過(guò)提取樣本DNA，用檢測(cè)部分D型的試劑盒對(duì)RHD基因10個(gè)外顯子進(jìn)行檢測(cè)，以確認(rèn)其實(shí)屬于包含所有10個(gè)外顯子的Rh(D)陽(yáng)性樣本。利用全血總RNA提取試劑盒從此臍血網(wǎng)織紅細(xì)胞中提取總RNA，根據(jù)設(shè)計(jì)的不同引物，分別采用一步法和兩步法進(jìn)行RT-PCR擴(kuò)增，通過(guò)瓊脂糖凝膠電泳分離目的基因片段，再切膠并溶膠純化目的基因；將純化后的基因片段連接至TOPO TA克隆測(cè)序載體pCR4-TOPO，通過(guò)轉(zhuǎn)化至TOP10大腸桿菌、以適量菌液均勻涂布至含氨芐青霉素的普通瓊脂平板，37℃倒置過(guò)夜培養(yǎng)，，其后，隨機(jī)挑取單一菌落至含氨芐青霉素的液體培養(yǎng)基中37℃水平震搖過(guò)夜增菌篩選培養(yǎng)，隨后以堿裂解法提取純化質(zhì)粒，再對(duì)提取的質(zhì)粒進(jìn)行測(cè)序，挑選出序列完整無(wú)突變的質(zhì)粒；以該篩選質(zhì)粒為模板，使用設(shè)計(jì)的新引物進(jìn)行PCR，擴(kuò)增為構(gòu)建表達(dá)載體用的目的基因，經(jīng)瓊脂糖凝膠電泳后切膠純化，再將其連接至真核表達(dá)載體pcDNA3.1/V5-His-TOPO，通過(guò)再次轉(zhuǎn)化至大腸桿菌、篩選增菌培養(yǎng)，提取并純化質(zhì)粒，最后經(jīng)質(zhì)粒測(cè)序篩選出含有序列正確、無(wú)突變目的基因，且插入方向正確的重組質(zhì)粒。 結(jié)果：血清學(xué)鹽水試管法初步檢測(cè)結(jié)果顯示2份樣本均為Rh(D)陽(yáng)性，Rh表型分別為DCcEe和DCCee；基因分型結(jié)果再次驗(yàn)證兩樣本均為Rh(D)陽(yáng)性樣本：沒(méi)有發(fā)生外顯子缺失。其后，成功提取樣本總RNA，一步法RT-PCR擴(kuò)增共獲得五個(gè)轉(zhuǎn)錄子條帶，分別為正常RHDcDNA，DEL9，DEL89，DEL79及DEL789。由于目的條帶較弱，隨后采用兩步法RT-PCR獲得正常RHDcDNA條帶，紫外燈下切膠后溶膠純化，而DEL9和DEL89轉(zhuǎn)錄子則直接合成。含上下游非編碼區(qū)的RHDcDNA成功克隆至TOPO TA克隆測(cè)序載體，經(jīng)過(guò)克隆篩選、質(zhì)粒序列分析結(jié)果顯示：獲得的片段中含有正常無(wú)突變RHD基因編碼區(qū)序列（包含基因上下游非編碼區(qū)的序列），以此含RHDcDNA的篩選質(zhì)粒為模板，經(jīng)PCR擴(kuò)增獲得從起始密碼子至終止密碼子的RHD基因編碼區(qū)全長(zhǎng)序列（不含上下游非編碼區(qū)）。之后將大量擴(kuò)增后獲得的RHDcDNA、以及DEL9和DEL89分別成功克隆至pcDNA3.1/V5-His-TOPO真核表達(dá)載體，經(jīng)過(guò)篩選克隆、測(cè)序驗(yàn)證，三種目的基因片段序列完整無(wú)突變且插入方向均正確，從而成功構(gòu)建3種不同RHD轉(zhuǎn)錄子的真核表達(dá)載體。 結(jié)論：實(shí)驗(yàn)成功構(gòu)建了正常RHDcDNA、以及DEL9和DEL89轉(zhuǎn)錄子的真核表達(dá)載體。RHD基因不同轉(zhuǎn)錄子表達(dá)載體的構(gòu)建為進(jìn)一步研究Rh(D)抗原的膜表達(dá)機(jī)理奠定了基礎(chǔ)。DEL9及DEL89轉(zhuǎn)錄子的表達(dá)載體構(gòu)建為進(jìn)一步研究D放散型（Del）個(gè)體紅細(xì)胞Rh(D)膜抗原表達(dá)機(jī)理奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R3416

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