本文選題：淋巴毒素　切入點(diǎn)：隨機(jī)突變　出處：《第二軍醫(yī)大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：淋巴毒素(Lymphotoxin α,簡(jiǎn)稱LT α,或LT)是由淋巴細(xì)胞在應(yīng)激情況下分泌的一種細(xì)胞因子。LT是TNF超家族的一個(gè)重要成員,LT與TNF超家族的另一成員TNF作用于兩個(gè)相同的膜受體,TNFRⅠ和TNFRⅡ,并擁有許多共同的生物學(xué)活性。LT通過(guò)TNFRI傳遞凋亡信號(hào),從而發(fā)揮抗腫瘤活性。 根據(jù)LT發(fā)揮生物學(xué)活性的特點(diǎn),我們嘗試通過(guò)增強(qiáng)LT與TNFRI的結(jié)合活性,達(dá)到增加LT抗腫瘤活性的目的。從LT與TNFRI受體的復(fù)合物晶體結(jié)構(gòu)數(shù)據(jù)可知,LT是通過(guò)某些特定氨基酸與TNFRI互相作用的。在此研究基礎(chǔ)上,本課題采用體外分子進(jìn)化策略,在LT的受體結(jié)合區(qū)域設(shè)計(jì)氨基酸隨機(jī)突變,建立重組人淋巴毒素(rhLT)的變異體庫(kù),并利用噬菌體展示技術(shù)進(jìn)行體外受體親和篩選,獲得與TNFRI受體結(jié)合力提高的rhLT突變體,進(jìn)一步研究其抗腫瘤等生物學(xué)活性。 一.新型噬菌粒展示載體的構(gòu)建以及功能蛋白的展示 構(gòu)建高效的噬菌體展示載體是突變體庫(kù)能夠成功展示和篩選的前提,而現(xiàn)有的pCANTAB5X還存在克隆位點(diǎn)少、展示外源多肽活性低、制備重組噬菌體庫(kù)庫(kù)容小以及展示大分子多肽受限制等問(wèn)題。為了解決以上問(wèn)題,我們將柔性多肽接頭和多克隆位點(diǎn)引入噬菌粒載體pCANTAB5X中。應(yīng)用5’端含Xba I、Stu I、Sal I、Kpn I識(shí)別序列的引物,PCR擴(kuò)增獲得Xba I-Stu I-Sal I-Kpn I-(G4S)3-Not I銜接片段,將該P(yáng)CR產(chǎn)物克隆到pMD-18T中,再將該片段插入pCANTAB5X中構(gòu)建成新型噬菌粒展示載體pCANTAB5L。限制性內(nèi)切酶酶譜及DNA序列分析證明(G4S)3多肽接頭和限制內(nèi)酶切位點(diǎn)Xba I、Stu I、Sal I、Kpn I序列被引入到新構(gòu)建的噬菌粒載體pCANTAB5L中。成功構(gòu)建了新型噬菌粒展示載體pCANTAB5L,并能有效展示功能靶蛋白、功能靶蛋白變異體庫(kù)和大分子量功能蛋白(300個(gè)氨基酸)。 進(jìn)一步改造噬菌體展示載體,在pCANTAB5S多克隆位點(diǎn)中引入SacI限制酶切位點(diǎn),并校正了pCANTAB5L載體Stu I、Sal I等位點(diǎn)的讀框,pCANTAB5S噬菌�？捎糜谥苽湔故疽吧蚉Ⅲ外殼蛋白的空白噬菌體。 二.重組人淋巴毒素噬菌體庫(kù)的構(gòu)建及受體親和篩選 構(gòu)建適合的大容量生物大分子變異體文庫(kù)是進(jìn)行體外分子進(jìn)化研究的關(guān)鍵,為了擴(kuò)大LT基因序列中被篩選的氨基酸范圍,我們采用含隨機(jī)核苷酸序列的引物,通過(guò)Overlap PCR的方法對(duì)LT的受體結(jié)合區(qū)域進(jìn)行多點(diǎn)隨機(jī)突變,分別構(gòu)建了rhLT R46+S106+L130三點(diǎn)隨機(jī)突變組合文庫(kù)和R46～A52、S106～F110、R46～A52+S106～F110區(qū)域隨機(jī)突變體庫(kù)。通過(guò)序列測(cè)定發(fā)現(xiàn),所構(gòu)建的點(diǎn)突變體文庫(kù)和區(qū)域突變體文庫(kù)在核苷酸和氨基酸的一級(jí)結(jié)構(gòu)上都具備了良好的隨機(jī)性和多樣性。對(duì)三點(diǎn)隨機(jī)突變組合文庫(kù)中30個(gè)樣品進(jìn)行原核表達(dá)和生物學(xué)活性測(cè)定,結(jié)果70%(21個(gè))的樣品無(wú)活性、23.3%(7個(gè))的樣品活性低于rhLT、6.7%(2個(gè))的樣品活性高于rhLT,具備了生物學(xué)活性的多樣性。 將rhLT突變體庫(kù)構(gòu)建于噬菌粒展示載體pCANTAB5L,獲得的rhLT重組噬菌體庫(kù)的滴度達(dá)到了10~(13)以上。噬菌體庫(kù)與固相化TNFR1受體進(jìn)行親和篩選,富集了能與TNFR1受體結(jié)合的rhLT突變體。隨機(jī)挑選20個(gè)單克隆噬菌體進(jìn)行ELISA受體結(jié)合鑒定,發(fā)現(xiàn)80%的克隆與TNFR1受體特異性結(jié)合,其中4個(gè)克隆與TNFR1受體的結(jié)合能力高于野生型序列的rhLT。 三.重組人淋巴毒素突變體的特性鑒定
[Abstract]:Lymphotoxin (Lymphotoxin alpha, alpha LT, or LT) is a cytokine secreted by.LT lymphocytes under stress conditions is an important member of the TNF superfamily, LT and TNF superfamily, another member of the TNF role in the two film of the same receptor, TNFR I and TNFR II, and hold the there are many common biological activity of.LT apoptosis signal transmission through TNFRI, so as to exert antitumor activity.
According to the characteristics of LT play biological activities, we try to enhance the binding activity of LT and TNFRI, increase the antitumor activity of LT to achieve from the crystal structure of the complex data of LT and TNFRI receptor that LT interaction through some specific amino acid and TNFRI. This research is based on the in vitro molecular evolutionary strategy in LT receptor binding domain of amino acid mutations were designed, the establishment of recombinant human lymphotoxin (rhLT) mutation libraries and using phage display technology in vitro receptor affinity screening, rhLT mutant and TNFRI receptor binding force increase, further research on its antitumor activity.
Construction of a new type of bacteriophage display carrier and display of functional protein
Construction of phage display vector, is a prerequisite for successful show and screening of mutant library, while the existing pCANTAB5X are cloning sites less, showing an exogenous polypeptide activity is low, the preparation of recombinant phage display library of small and large peptides is limited. In order to solve the above problems, we will be flexible joint and the multiple cloning sites of polypeptide the phagemid vector pCANTAB5X. The application of the 5 'end with Xba I, Stu I, Sal I, Kpn primer I recognition sequence, PCR I-Stu I-Sal I-Kpn I- amplified Xba (G4S) 3-Not I connection fragment, the PCR product was cloned into pMD-18T, then the fragment was inserted into pCANTAB5X to construct model phagemid vector pCANTAB5L. restriction endonuclease enzyme and sequence analysis showed that the DNA (G4S) 3 peptide and restriction digestion sites of Xba joint I, Stu I, Sal I, Kpn I sequence was introduced into the phagemid vector pCANTAB to construct the new In the 5L, we successfully constructed a new phage display vector pCANTAB5L, and effectively displayed functional target protein, functional target protein variant library and macromolecular functional protein (300 amino acids).
We further modified phage display vector, introduced SacI restriction enzyme site in pCANTAB5S polyclonal site, and corrected the reading frame of pCANTAB5L carrier Stu I, Sal I and other loci. PCANTAB5S phagocytin can be used to prepare the blank phage displaying wild type P III coat protein.
Two. Construction of recombinant human lymphotoxin phage library and its receptor affinity screening
Construction of large molecule mutation library is the key for the study of molecular evolution in vitro, in order to expand the scope of the amino acid sequences of LT gene were selected, we use random primers containing nucleotide sequences, by means of Overlap PCR on LT receptor binding domain of multi point random mutation were constructed with rhLT R46+S106+L130 three random mutant library and R46 ~ A52, S106 ~ F110, R46 ~ A52+S106 ~ F110 regional random mutant library by sequencing. Found that the point mutant library and regional mutant libraries have randomness and good diversity in the primary structure of nucleotide and amino acids of three points. A sample of 30 random mutation combinatorial library in prokaryotic expression and biological activity determination results, 70% (21) were inactive, 23.3% (7) of the samples was lower than that of rhLT, 6.7% (2) The activity of the sample is higher than that of rhLT, and has the diversity of biological activity.
The rhLT mutant library constructed in phage display vector pCANTAB5L, the recombinant rhLT phage library titer obtained was 10~ (13). Phage library with solid TNFR1 receptor affinity screening rhLT mutant enriched binds to TNFR1 receptor. Randomly selected 20 single clone phage ELISA receptor identification. Found that 80% clones with TNFR1 receptor specific binding, the binding capacity of 4 clones with TNFR1 receptor is higher than that of the wild type sequence rhLT.
Three. Characterization of recombinant human lymphotoxin mutants

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392.1

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相關(guān)期刊論文 前1條

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