Introduction

In order to use photo-inactivated Leishmania as a safe carrier to deliver specific antigens to the APCs for T and B cell stimulation, we designed an E. coli-Leishmania shuttle vector for antigen expression in Leishmania.

A shuttle vector is a vector constructed so that it can reproduce in two different host species. The main purpose of these vectors is that they can be quickly amplified in E. coli and then manipulated in another organism, such as Leishmania. Here we designed an E.coli-Leishmania shuttle vector constructed under biobrick standards to provide a standardized shuttle vector for our own experiment and for others' future application.

Design

According to Dr. Kwang-Poo Chang’ s research , we found an interesting sequence in leishmania genome, which contains two coding regions, p36 and NAGT. These two coding regions are regulated by their own 5’UTR and 3’UTR, between the p36 and NAGT region is a 2300 bp intrinsic sequence, which is composed of the 3’UTR of p36 and the 5’UTR of NAGT. The 2300 bp intrinsic sequence has several stage-independent splicing sites, and allowed the genes to be constitutively expressed in any stage of leishmania. According to its features, we decided to make it a leishmania shuttle vector which could be used in biobrick form.

The first coding region, p36, is replaced with hygromycin resistant gene as a selection marker in leishmania, and combined with 5’UTR as a biobrick part, since the 5’UTR may contains promoter and ribosome binding site and other important functions in leishmania. As for the second coding region, NAGT, is allowed to be replaced with any protein we want leishmania to carry. In our project, we want to put hemagglutinin (HA) of H1N1 and ovalbumin (OVA) into the site to prove our concept. The 3’UTR of the sequence was designed to become a terminator part while the 2300 intrinsic sequence was also needed to regulate the expression of the protein in the shuttle vector.

The leishmania gene would be put into pSB1C3 to become a shuttle vector that could quickly proliferate in e.coli and express proteins in leishmania. There are 3 advantages of our shuttle vector, the first is that it contains two coding regions originally, so our shuttle vector will be a good choice to express two proteins at the same time. Second, the 2300 intrinsic sequence in our shuttle vector has many stage-independent splicing sites to cut the polycistronic RNA into two transcripts in any stage of leishmania. The third is that, through the biobrick design, we can build a standard shuttle vector for leishmania and also introduce a new animal model into iGEM competition.

Results

The construction of pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR
We synthesized the 5’HYG, 3’UTR, HA, OVA sequence directly by IDT. The synthesized sequence were digested and ligated to pSB1C3. The parts were checked by PCR. The length of 5’HYG was 1446 bp, HA was 1700 bp, OVA was 2098 bp and 3’UTR was 774 bp (Fig. 1, lane A.B.C.D). To build a workable leishmania shuttle vector, we generate two constructs, pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR (Fig. 1, lane F.G.H.I). The plasmids were purified by midiprep, and transfected into leishmania by electroporation.

(Fig. 1) All the parts in our shuttle vector were checked by PCR
We checked all the biobrick parts used in our construct by PCR. The forward primer sequence was 5’- GAATTCGCGGCCGCTTCTAGAG-3’, which was in the prefix site. And the reverse primer sequence was 5’-CTGCAGCGGCCGCTACTAGTA-3’, which was in the suffix site. The reaction was done by Taq polymerase and screened in 0.8％ agarose gel by electrophoresis. The construction of 2300 intron had some problems, so its length was about 2~2.1 kb (lane E). As for the composite parts, the HA-3’UTR should be 2.6 kb in length, while the OVA-3’UTR should be 2.9 kb in length.After the ligation with 5’HYG, the length of the 5’HYG-HA-3’UTR was about 4.1 kb, and the 5’HYG-OVA-3’UTR should be 4.5 kb in length.

The construction of pSB1C3-2300 intron

Since the 2300 intron contained too many CG pairs, it’s not allowed to synthesis. We used point mutation to change the nucleotide in the 2300 bp sequence, therefore, the sequence would be separated into 3 parts. Through the PCR, we could have these 3 parts amplified from p6.5 plasmid, then used the PCR-after-ligation strategy to construct the pSB1C3-2300intron part (Fig. 1, lane E). The reason why we used the PCR-after-ligation strategy was because the ligation rate of the sequence was really low. However, although the parts of 2300 intron could be proliferated by PCR, we were unable to ligate the 3 parts together. The sequencing results of the 2300 intron always lost the second part, no matter what strategy we used in the construction. So, it turned out that we couldn’t put the 2300 intrinsic region into the final construction of our shuttle vector.

Construction of pSB1C3-5’HYG-GFP-3’UTR

Since we can’t detect the HA and OVA protein by western blotting after the pSB1C3-5’HYG-HA-3’UTR and pSB1C3-5’HYG-OVA-3’UTR plasmid were transfected into leishmania. We decided to construct pSB1C3-5’HYG-GFP-3’UTR in order to prove if our leishmania shuttle vector could express the GFP or not. The GFP sequence came from BBa_E0040 in the vector pSB1A2.

The right colony of pSB1C3-5’HYG-GFP-3’UTR was picked and amplified in 200 ml LB broth, then the plasmid DNA was purified by midiprep and transfected into leishmania by electroporation.

(Fig. 6 ) pSB1C3-GFP-3’UTR and pSB1C3-5’HYG-GFP-3’UTR check by colony PCR
The PCR was performed with Taq polymerase, and screened in 0.8％ agarose gel by electrophoresis. The GFP-3’UTR was about 1.5 kb (720 bp +774 bp) in length, and the 5’HYG-GFP-3’UTR was about 3 kb (1446 bp +720 bp +774 bp).

Introduction

To prove our concept, we tested the efficiency of the antibody immune response and T cell immune response of the photo-inactivated Leishmania as a vaccine adjuvant. Ovalbumin (OVA) has been commonly used as the antigen for testing the efficiency of antibody response and T cell activation in previous immunology experiments. Also, OVA is the only foreign antigen carried by Leishmania that has been shown to load the major histocompatibility complex class I molecules (MHC I) after phagocytosis by APCs, since the transgenic mutants of Leishmania is a new way to deliver antigens into antigen-presenting cells (APC). Thus, we want to use OVA in our in vivo test to validate our hypothesis of photo-inactivated Leishmania as an adjuvant. We co-injected OVA recombinant protein and photo-inactivated Leishmania that is genetically modified to present OVA protein into mouse.

Serum is collected every 5 days after the second injection to test the antibody immune response with Anti-OVA ELISA and further tested the T cell response with dissected splenic cell. The outcome will be compare to the of Alum adjuvant.

Design

We subcutaneously co-injected leish-OVA (Leishmania expressing OVA) and OVA protein into mice and compare the outcome to the of Alum adjuvant with OVA protein as a positive control. We immunized the mice twice, the second boost will be injected on the 15th day after the first shot and after the second shot we will collect serum from the mice on the 5th 10th 13th day after. The serum will be tested for anti-OVA IgG1 and IgG2a as the antibody response and the cell-mediated immune response, respectively. We will dissect the spleen on the 10th day after the second boost and culture the splenic cells for 6 days. Culture supernatant will be tested for cytokines specific for T cell response.

Results

In order to use photo-inactivated Leishmania as a safe carrier to deliver specific antigens to the APCs for T and B cell stimulation, we designed an E. coli-Leishmania shuttle vector for antigen expression in Leishmania.

A shuttle vector is a vector constructed so that it can reproduce in two different host species. The main purpose of these vectors is that they can be quickly amplified in E. coli and then manipulated in another organism, such as Leishmania. Here we designed an E.coli-Leishmania shuttle vector constructed under biobrick standards to provide a standardized shuttle vector for our own experiment and for others' future application.

Discussion

In order to use photo-inactivated Leishmania as a safe carrier to deliver specific antigens to the APCs for T and B cell stimulation, we designed an E. coli-Leishmania shuttle vector for antigen expression in Leishmania.

A shuttle vector is a vector constructed so that it can reproduce in two different host species. The main purpose of these vectors is that they can be quickly amplified in E. coli and then manipulated in another organism, such as Leishmania. Here we designed an E.coli-Leishmania shuttle vector constructed under biobrick standards to provide a standardized shuttle vector for our own experiment and for others' future application.

Introduction

The biobrick construct of E.coli-Leishmania shuttle vector is meant to express the targeting antigen protein in Leishmania through amplification in E.coli and transfection into Leishmania. Therefore, total size of the shuttle vector can significantly affect the efficiency of transformation and transfection during the procedure. To enhance the efficiency, we've tried to focus on shortening the targeting antigen sequence which will then be sub-cloned into the shuttle vector. In order to identify antigen sequence with the highest MHC binding affinity, researchers have to utilize several bioinformatics tools to figure out or predict the protein properties. In our project, we have generated an integrated protein information website, McHug, to help users in searching for peptide sequences that can optimally activate immune response. Other than providing users with all basic protein information, McHug features the visualized interface which can transform loads of numerical data into legible charts. The ultimate goal of McHug website is to mark all the protein annotations on the given protein sequence and display the relative immune properties such as MHC binding affinity in every position of the protein. Selection of the most suitable sequence for MHC presentation can be easily accomplished with McHug. The apllicability of McHug website was further tested in vitro using OVA-loaded dendritic cells. MHC molecules were immunoprecipitated and the associated peptide sequences were then analyzed by mass spectrometry. You can learn faster and more about your targeting antigen while experiencing McHug.

Design

In order to use photo-inactivated Leishmania as a safe carrier to deliver specific antigens to the APCs for T and B cell stimulation, we designed an E. coli-Leishmania shuttle vector for antigen expression in Leishmania.

A shuttle vector is a vector constructed so that it can reproduce in two different host species. The main purpose of these vectors is that they can be quickly amplified in E. coli and then manipulated in another organism, such as Leishmania. Here we designed an E.coli-Leishmania shuttle vector constructed under biobrick standards to provide a standardized shuttle vector for our own experiment and for others' future application.

Results

In order to use photo-inactivated Leishmania as a safe carrier to deliver specific antigens to the APCs for T and B cell stimulation, we designed an E. coli-Leishmania shuttle vector for antigen expression in Leishmania.

A shuttle vector is a vector constructed so that it can reproduce in two different host species. The main purpose of these vectors is that they can be quickly amplified in E. coli and then manipulated in another organism, such as Leishmania. Here we designed an E.coli-Leishmania shuttle vector constructed under biobrick standards to provide a standardized shuttle vector for our own experiment and for others' future application.

Discussion

In order to use photo-inactivated Leishmania as a safe carrier to deliver specific antigens to the APCs for T and B cell stimulation, we designed an E. coli-Leishmania shuttle vector for antigen expression in Leishmania.

A shuttle vector is a vector constructed so that it can reproduce in two different host species. The main purpose of these vectors is that they can be quickly amplified in E. coli and then manipulated in another organism, such as Leishmania. Here we designed an E.coli-Leishmania shuttle vector constructed under biobrick standards to provide a standardized shuttle vector for our own experiment and for others' future application.