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摘要：变异型兔出血症病毒（Rabbit hemorrhagic disease virus variant，RHDV2）VP60蛋白是免疫保护性抗原，在诱导抗病毒感染的免疫反应中发挥重要作用。本实验以质粒pMD19-T-VP60-2为模板，PCR扩增获取VP60基因的主要抗原序列。然后用DNase I随机消化PCR产物，T4 DNA聚合酶补平，连接pEcoR I Linker和EcoR I酶切，以获取EcoR I识别的黏性末端。同时用pEcoR I酶切pC89 pⅧ型噬菌粒载体并进行去磷酸化处理。Microcon超滤装置回收20-125bp带有黏性末端的随机片段，并与pC89噬菌粒载体进行连接。将重组噬菌粒转化XL1-Blue感受态细胞，用辅助噬菌体VCSM13超感染，使制备的随机片段以融合蛋白形式展示于噬菌体表面，从而构建了RHDV2基因特异性噬菌体展示肽库。测定肽库滴度。结果显示所建肽库滴度约为3.5×1012PFU/mL，可以满足VP60抗原表位的筛选。27471
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毕业论文关键词：变异型兔出血症病毒；VP60基因；特异性噬菌体展示肽库
Construction of VP60 Gene - specific Phage Peptide Library for Rabbit Hemorrhagic Disease Virus Variant(RHDV2)
Abstract：Variant Rabbit hemorrhagic disease virus (RHDV2) VP60 protein is an immune protective antigen that plays an important role in antiviral infection. In this study, VP60 gene was amplified by PCR using plasmid pMD19-T-VP60-2 as template. The PCR product was randomly digested by DNase I, catalyzed by T4 DNA polymerase to generate blunt ends, ligated with pEcoR I Linker and followed by EcoR I digestion to obtain the EcoR I–recognized viscous ends. The pC89-pVIII phagemid vector was digested with pEcoR I and subjected to dephosphorylation. The random fragments of 20-125bp with sticky ends were purified by the Microcon ultrafiltration device and were inserted into the pC89 phagemid vector. The recombinant phagemids was transformed into XL1-Blue competent cells and superimposed with the helper phage VCSM13. Obtained random fragments were expressed on the phage surface in form of fusion proteins, and thus the RHDV2 gene specific phage display peptide library was constructed. The peptide titer was determined. The results showed that the titer of the peptides was about 3.5×1012PFU/mL, which could be used to screen the VP60 epitopes.
Key words: RHDV2；VP60；Gene-specific phage peptide library
目 录
摘要.1
关键词..1
Abstract1
Key words..1
引言.1
1 材料与方法.2
1.1 材料..2
1.1.1 质粒、菌株、噬菌粒及辅助噬菌体2
1.1.2 主要试剂 ..2
1.2 VP60基因的扩增.2
1.3 琼脂糖凝胶电泳检测VP60扩增效果3
1.4 VP60扩增产物的回收. ..3
1.5 VP60 基因随机消化.. ..3
1.5.1 VP60 PCR产物预处理. 3
1.5.2 DNaseⅠ消化条件的优化.3
1.5.3 VP60基因随机消化 3
1.5.4 消化酶切片段的纯化回收.. .4
1.6 VP60消化酶切片段的补平. .4
1.6.1 补平4
1.6.2 补平片段的纯化回收.4
1.7 VP60补平片段的去磷酸化 ..4
1.8 VP60去磷酸化片段加接头 ..4
1.9 VP60加接头片段酶切.. .4
1.9.1 酶切. ..4
1.9.2 VP60加接头片段酶切.4
1.10 噬菌粒载体的制备. 4
1.11 酶切片段VP60与噬菌粒载体PC89的连接 .5
1.12 高效感受态宿主菌的制备. 6
1.12.1 高效感受态宿主菌敏感性鉴定.6
1.13 辅助噬菌体VCSM13的制备.6
1.13.1 辅助噬菌体VCSM13的复苏.. 6
1.13.2 辅助噬菌体VCSM13的扩增. .6
1.13.3 辅助噬菌体VCSM13滴度的测定. 6
1.14 VP60基因特异性噬菌体展示肽库的构建. 6
1.15 VP60基因特异性噬菌体展示肽库滴度的测定 7
2 结果与分析. 7
2.1 VP60基因PCR扩增与测序..7