重组人淋巴毒素突变体库的构建及受体亲和筛选

发布时间：2018-03-02 19:31

本文选题：淋巴毒素　切入点：随机突变　出处：《第二军医大学》2005年硕士论文　论文类型：学位论文

【摘要】：淋巴毒素(Lymphotoxin α,简称LT α,或LT)是由淋巴细胞在应激情况下分泌的一种细胞因子。LT是TNF超家族的一个重要成员,LT与TNF超家族的另一成员TNF作用于两个相同的膜受体,TNFRⅠ和TNFRⅡ,并拥有许多共同的生物学活性。LT通过TNFRI传递凋亡信号,从而发挥抗肿瘤活性。根据LT发挥生物学活性的特点,我们尝试通过增强LT与TNFRI的结合活性,达到增加LT抗肿瘤活性的目的。从LT与TNFRI受体的复合物晶体结构数据可知,LT是通过某些特定氨基酸与TNFRI互相作用的。在此研究基础上,本课题采用体外分子进化策略,在LT的受体结合区域设计氨基酸随机突变,建立重组人淋巴毒素(rhLT)的变异体库,并利用噬菌体展示技术进行体外受体亲和筛选,获得与TNFRI受体结合力提高的rhLT突变体,进一步研究其抗肿瘤等生物学活性。一.新型噬菌粒展示载体的构建以及功能蛋白的展示构建高效的噬菌体展示载体是突变体库能够成功展示和筛选的前提,而现有的pCANTAB5X还存在克隆位点少、展示外源多肽活性低、制备重组噬菌体库库容小以及展示大分子多肽受限制等问题。为了解决以上问题,我们将柔性多肽接头和多克隆位点引入噬菌粒载体pCANTAB5X中。应用5’端含Xba I、Stu I、Sal I、Kpn I识别序列的引物,PCR扩增获得Xba I-Stu I-Sal I-Kpn I-(G4S)3-Not I衔接片段,将该PCR产物克隆到pMD-18T中,再将该片段插入pCANTAB5X中构建成新型噬菌粒展示载体pCANTAB5L。限制性内切酶酶谱及DNA序列分析证明(G4S)3多肽接头和限制内酶切位点Xba I、Stu I、Sal I、Kpn I序列被引入到新构建的噬菌粒载体pCANTAB5L中。成功构建了新型噬菌粒展示载体pCANTAB5L,并能有效展示功能靶蛋白、功能靶蛋白变异体库和大分子量功能蛋白(300个氨基酸)。进一步改造噬菌体展示载体,在pCANTAB5S多克隆位点中引入SacI限制酶切位点,并校正了pCANTAB5L载体Stu I、Sal I等位点的读框,pCANTAB5S噬菌粒可用于制备展示野生型PⅢ外壳蛋白的空白噬菌体。二.重组人淋巴毒素噬菌体库的构建及受体亲和筛选构建适合的大容量生物大分子变异体文库是进行体外分子进化研究的关键,为了扩大LT基因序列中被筛选的氨基酸范围,我们采用含随机核苷酸序列的引物,通过Overlap PCR的方法对LT的受体结合区域进行多点随机突变,分别构建了rhLT R46+S106+L130三点随机突变组合文库和R46～A52、S106～F110、R46～A52+S106～F110区域随机突变体库。通过序列测定发现,所构建的点突变体文库和区域突变体文库在核苷酸和氨基酸的一级结构上都具备了良好的随机性和多样性。对三点随机突变组合文库中30个样品进行原核表达和生物学活性测定,结果70%(21个)的样品无活性、23.3%(7个)的样品活性低于rhLT、6.7%(2个)的样品活性高于rhLT,具备了生物学活性的多样性。将rhLT突变体库构建于噬菌粒展示载体pCANTAB5L,获得的rhLT重组噬菌体库的滴度达到了10~(13)以上。噬菌体库与固相化TNFR1受体进行亲和筛选,富集了能与TNFR1受体结合的rhLT突变体。随机挑选20个单克隆噬菌体进行ELISA受体结合鉴定,发现80%的克隆与TNFR1受体特异性结合,其中4个克隆与TNFR1受体的结合能力高于野生型序列的rhLT。三.重组人淋巴毒素突变体的特性鉴定
[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

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392.1

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