Cloning and expression of p32 gene of sheep poxvirus and its preparation of monoclonal antibody

目录

Abstract

摘要

LIST OF CONTENTS

LIST OF ABBREVIATIONS

Review Part Sheep and goat pox

1．Deftnition

2．Etiology

3．Epidemiology

4．Host range

5．Clinical signs

6．Routes of transmission

6．1．Direct transmission

6．2．Indirect transmission

7．Virus infection and replication

8．Pathogenesis

9．Diagnosis

9．1．Polymerase chain reaction (PCR)

9．2．Enzyme linked immune sorbent assay (ELISA)

9．3．Western blotting

10．Virus isolation

11．Treatment

12．Control and prevention

13．Objectives for this study

Research Part Ⅰ Cloning and expression of p32 gene of sheep poxvirus

1．1．1 Main materials

1．1．2 Mice

1．1．3 Primers and PCR system

1．1．4 Recovery of the PCR fragments

1．1．5 Digestion and ligation

1．1．6 Transformation into the DH5α

1．1．7 Identification of the recombinant plasmids

1．1．8 Expression of p32 protein induced by IPTG

1．1．10 Western blotting analysis for the expression of GST-P32

1．1．11 Purification and identification of recombinant proteins

1．1．12 Preparation of polyclonal antibodies against P32

1．1．13 IFA analysis for mouse polyelonal antibody against P32

1．1．14 Western blot analysis for mouse polyelonal antibody against P32

1．2 Results

1．2．1 Construction and identification of the recombinant plasmid pGEX-6p-1-P32 and pcDNA3．1-P32

1．2．2 Identification of the expression and purification of GST-P32 fusion protein

1．2．3 Identification of polyclonal antibodies against P32

1．3 Discussion

Part Ⅱ：Development monoclonal antibodies against P32 protein of sheep poxvirus

2．1 Materials and Methods

2．1．1 Main materials

2．1．2 A peptide-based ELISA for detection of antibody against P32

2．1．3 Immunized mice

2．1．4 Cell fusion

2．1．5 Screening of positive hybridoma cells by peptide-based ELISA

2．1．6 Identification of the subclasses of monoclonal antibody

2．1．7 Monocional antibodies against P32 were confirmed by IFA

2．1．8 Monoclonal antibodies against P32 were confirmed by Western blot

2．1．9 Identification of antigenic epitopes recognized by mAbs against P32

2．2 Results

2．2．1 Development of a peptide-based ELISA

2．2．2 Screening monoclonal antibodies against P32 by the peptide-based ELISA

2．2．3 mAbs 1D5 and 3A6 efficiently recognized the P32 protein expressed in DF-1

2．2．4 Identification of antigenic epitopes against P32 mAb

2．3 Discussion

SUMMARY

REFERENCES

Acknowledgements

声明