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An Ag43Expressing System Used for Bacterial Surface Display of Chimeric Recombinant Protein as Vaccine

Author: LiYueNan
Tutor: ZhouPeng
School: Hainan University
Course: Agricultural biotechnology
Keywords: Bacterial surface antigen43(Ag43) Immune tolerance Geneengineering expression system Endoglin Recombinant protein vaccine Immunotherapy Tumor angiogenesis
CLC: S852.52
Type: PhD thesis
Year: 2012
Downloads: 78
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Abstract


Nowaday, vaccine has become the most effective approach for controlling human and animal infectious diseases. As the advance in biomedical studies, more and more pathogens that cause human diseases are found from the animal hosts. The most notorious example is the human immunodeficiency virus (HIV), which are though to host in human close-relative, African green monkey. Recent worldwide pandemic of Severe Acute Respiratory Syndromes (SARS) and avian influenza let human learn more about human-animal relationship. Thus, the techniques that human invent to prepare any kinds of vaccines for human disease-control can be widely adapted to prepare animal vaccines, including agriculture animals (such as domestic animals, poultries and pets). Animal vaccines not only can prepvent animal diseases, but also prevent human from the pathogens that are originally hosted in animals. Not just for prevention of infectious diseases in human and animals, vaccine now can even be used to prevent and treat cancer, the first human killer in the world. In general, all approaches that human invent to prepare vaccines for controlling human diseases can be used to prepare vaccine for agriculture animals.Previous vaccine studies and some adjuvant contained ingredients of bacteria antigens, such as mycobacterium tuberculosis is one of necessary ingredients in the common-used Freund’s complete adjuvant. Cholera toxin domain β combined with self-antigen has been widely used as vaccine to induce immune response against self immune tolerance. Bacterial antigen43(Ag43) is a surface antigen expressed by Escherichia coli K12strains and possess strong antigenicity, which can induce strong immune response. Bioinformatics analyses indicate that Ag43has various T and B epitopes which will facilitate induction of breaking immune tolerance against self antigen just as cholera toxin domain β. However, Ag43is different from cholera toxin domain β, it can independently express on the surface not depend on other auxiliary molecules (such as chaperone). In addition, Ag43can be easy isolated from the bacterial surface only through heating to about60℃, which is very convenient for vaccine preparation. Therefore, in this study, we hypothesize that Ag43can be used as a tool to prepare chimeric protein vaccines which express foreign antigen with Ag43 and display on the bacterial surface in assistant of the Ag43. The Ag43chemeric protein vaccine can be isolated by only heating that made it easy to prepare vaccine.In this study, Ag43gene in Escherichia coli JM109strain was knockouted by a special technique with the TargeTron Gene Knockout System, which led to disexpression of Ag43in the knockout bacteria (named as Tan109). Thereafter, a recombinant plasmid in which the Ag43gene was inserted into scaffold plasmid pET-22b was constructed by the standard gene engineering technology. This newly constructed recombinant was named as pETAg43, in which the Ag43gene contained all the signal peptide and β domain, but only partial a domain. The Ag43gene knockout bacteria strain Tan109and the recombinant plasmid pETAg43were identified by restriction enzyme analysis, PCR and gene sequencing. Therefore, the bacteria strain Tanl09and the recombinant plasmid pETAg43consist a prokaryotic expression system (named as Ag43bacterial surface display system). We used IPTG to induce pETAg43expression in Tan109, and the results indicated that the Ag43bacterial surface display system is a high throughput expression system.In order to know whether the Ag43bacterial surface display system is effective display Ag43chimeric protein on the bacterial surface, we used green fluorescin GFP as report marker to observe on the bacterial surface after the induction expression of GFP in the bacterial Tan109. We first amplified the GFP gene from a commercial plasmid pEGFP-C1, and inserted the amplified GFP gene into our recombinant plasmid pETAg43and gave a new recombinant plasmid pETAg43-GFP. After the plasmid pETAg43-GFP was proved to be correctly constructed by restriction enzyme analysis and gene sequencing, we transfected it into Tan109and induced expression by IPTG. Under fluorescence microscope, strong fluorescent signal was observed in the bacterial Tanl09transfected with the plasmid pETAg43-GFP, but not in the bacterial Tanl09not transfected with the plasmid pETAg43-GFP. In addition, we found that the fluorescent signal on bacterial Tanl09transfected with the plasmid pETAg43-GFP was almost disappeared when heated to about60℃, compared with the bacterial Tanl09not transfected with the plasmid pETAg43-GFP. These results indicated that the chemeric protein was indeed expressed on the bacterial surface. Moreover, we still investigated the optimum condition for isolation and preparation of the Ag43/GFP chimeric protein by heating method, and we found that the optimum temperature was not60℃, but55℃, and the optimum time of heating was40-60minutes. GFP is only a report protein that made us easy to observe the surface-display effects of the Ag43bacterial surface display system. The major aim of the present study is to preparation of Ag43chimeric protein as vaccine to induce breaking of immune tolerance against self-antigen. Thus, we used endoglin, a marker molecule of tumor angiogenesis and target of many antitumor therapeutic approaches, as model antigen to further study its possibility of preparing recombinant protein vaccine. As we did to construct plasmid pETAg43-GFP, the endoglin gene amplified from a ready-constructed plasmid in our previous study was inserted into the plasmid pETAg43, and gave a new recombinant plasmid pETAg43-END. After plasmid pETAg43-END was also proved to be correctly constructed by restriction enzyme analysis and gene sequencing, plasmid pETAg43-END was transfected into bacteria Tan109to induce expression on bacterial surface by IPTG. We prove the chimeric protein Ag43/END was displayed on the bacterial surface through observation of fluorescent signal induced by a monoclonal antibody against endoglin and a FITC-conjugated second antibody. Thereafter, the chimeric protein Ag43/END was isolated by heating to50℃for50minutes. SDS/PAGE electrophoresis indicated that the chimeric protein Ag43/END isolated by heating was very pure and showed only single band on the SDS/PAGE gel.We next used chimeric protein Ag43/END as a protein vaccine to observe its protective and therapeutic antitumor activities in three solid (CT26, Meth A and LL/2) and a metastatic (B16) tumor models. Our results showed that the tumor volume was significantly smaller and the survival rate was longer in the mice treated with Ag43/END vaccine, when compared with the tumor volume and the survival rates in the mice treated with control vaccines or vaccine vehicle. Statistical significance was existed in the Ag43/END vaccinated mice. Similar results were found in the B16metastatic model, the mice vaccinated with Ag43/END vaccine had few metastatic masses on the lung surface when compared with the control mice. In addition, the lung weigh in the mice vaccinated with Ag43/END was significantly heavy than that found in the control mice. It was unbelievable that we found the tumor mass in one mouse in the CT26model was completely disappeared after Ag43/END vaccination, leaving only scar in the tumor site.At last, we investigated the possible mechanisms by which Ag43/END vaccine induced antitumor activities. Western blot analysis showed that anti-endoglin antibodies were found in the serum from the mice vaccinated with Ag43/END vaccine, but not in the mice vaccinated with control vaccines or vehicle. Immunofluorescence detection of the tumor tissues with FITC-conjugated anti-mouse IgG antibody showed that significant fluorescent signal only found on the tumor tissues from the mice vaccinated with Ag43/END vaccine, the tumor tissues from the control mice show little fluorescence when compared with Ag43/END-vaccinated mice. The morphology of the fluorescent signal in the tumor tissues from the mice vaccinated with Ag43/END was in a vessel-like form, suggesting blood vessels. In addition, special B lymphocytes secreted antibodies against self endoglin were found in the spleen from the mice vaccinated with Ag43/END vaccine, not in the control mice. These results indicated that chimeric protein Ag43/END as vaccine could induce special immune response against self endoglin and produce anti-endoglin antibodies. Moreover, we observed the blood vessels by immunohistochemistry showed that the blood vessel density in the tumor tissues from the mice vaccinated with Ag43/END was significantly decreased, when compared with the control mice. Quantitative analysis of vessel generation by an alginate encapsulated tumor cell assay also indicated that the vessel generation in the Ag43/END-vaccinated mice was significantly reduced, when compared with the control mice.In summary, we had constructed a prokaryotic expression system (Ag43bacterial surface display system) that could express Ag43chimeric protein (such as GFP and endoglin) and displayed on the surface of host bacteria. The Ag43chimeric protein could be easy isolated from the bacteria surface by heating to about55℃. An Ag43and endoglin chimeric protein (Ag43/END) was an effective tumor model vaccine that could break immune tolerance against self endoglin and induced generation of anti-endoglin antibodies. It was the self anti-endoglin that caused decreased blood vessels in the tumor tissues, leading to inhibition of tumor growth in the mice vaccinated with Ag43/END as vaccine. These results suggest that Ag43bacterial surface display system is an excellent tool for development of vaccine that may be used to prepare agriculture vaccine for disease controlling in domestic animals, poultries and pets.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Serological > The immunoassay prevention ( vaccination )
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