Simplifying the RP4 plasmid

For example the IncPa plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins,[2] both of which contribute to Mpf.

Our aim is get a simpler conjugation plasmid. So we will choose another plasmid in IncPa .After getting the tra1 aPnd tra2 part from the plasmid RP4,we can ligate the two parts with the new plasmid backbone.

Replace the plasmid backbone

As described in the project page, we have successfully constructed the double plasmid system in the E.coli. Then we have tested our plasmid, and the mini plasmid works well. However we hope to find the most suitable plasmid backbone, it has a Broad-host-rang.

The following is our requirements:

First, because of the plasmid incompatibility, we want to use more incompatible plasmid to replace the original mini plasmid backbone.

Second, the plasmid must be high copy.

Third, the plasmid backbone is short.

Finally, It has reporter gene.

When we get a suitable plasmid backbone, we will do more tests and verify its conjugation capability.

Conjugating various types of recipient bacterium

In our project, the conjugation of recipient bacteria is a very important part. We plan to try more recipient. For example, we have made conjugation test between E.coli and E.coli ,E.coli and Vibrio harveyi.

We consider a Broad-host-rang is very significant for future application, such as environment and healthy.

In the future , we will try many Gram-negative, After test, we can get more data and have a very good analysis of conjugation. Finally we will make a list about the host rang.

Oral DNA vaccine

WHAT’S DNA VACCINE?

DNA vaccine is also called the nucleic acid vaccine or gene vaccine.It's an eukaryotic expression plasmid DNA(sometimes RNA) which codes immunogen or associated with immunogen. It can enter the animal’s cells through a certain way. After transfecting into the host cell, it will be translated and will express the antigen protein which can stimulate the body to produce two kinds of immune response including nonspecific immune and specific immune response. There by the immune protection takes place. The vaccine has both the advantage of attenuated vaccine and no risk of the opposite at the same time. Because of this the vaccine has got people's attention recently and it is seen as the third generation of vaccine following the traditional vaccine and genetically engineered subunit vaccine.

THE ADVANTAGE OF DNA VACCINES

Plasmid is very stable, easy to be stored and transported, easy to be prepared and used, easy for mass production by a low cost. Gene vaccine preparation is relatively safe and much easier compared with other vaccines, which are toxic and difficult to extract antigen. Plasmid DNA in the host can exist for a long time, antigen expression of genes in the body to produce antigen protein, constantly stimulate the body's immune system to produce long-term immunitye, and the immune effects are reliable. Gene vaccine can not only produce the humoral immune response, but can also lead to cytotoxic T lymphocyte activation and induce cellular immunity. But traditional vaccines only induce cellular immunity by live planting , and it has the danger of the vaccine's virulence picks upPreparation with core protein conservative DNA sequence of gene vaccine against pathogens (bacteria or viruses) of each variant subtypes can produce an immune response, so it can avoid the immune escape caused by pathogens variation.

A plasmid vector can be multiple antigen gene cloning of multivalent seedlings, thus many diseases can be prevented by only one kind of gene vaccine.

Plasmid DNA has no immunogenicity, which is different with recombinant vaccines that can induce immune response to the carrier, no anti DNA antibody has been reported at least.

THE APPLICATION OF DNA VACCINES.

DNA vaccine application field is very wide, it has been used in at least ten kinds of infectious disease prevention experiments, including viruses, bacteria and parasite adapting just-in-time inventory of various type of microorganisms. Some DNA vaccine are also being used on the part of the treatment of infectious diseases. In addition, it can also be used in the prevention or treatment of certain cancers. These disease include: hepatitis B (state-run), Influenza virus, human immunodeficiency virus (HIV), n/med tuberculosis bacterium, etc.

DNA vaccine as a new type of vaccine has promising prospects in the future. Using TAT:H4 as a transport device can improve the transfection efficiency and the efficiency of DNA vaccine. We can replace the EGFP with antigen gene of some fish disease to prevent fish from falling ill.

IN THE FUTURE

We will do these jobs:

1. Ichthyophthiriasis is a typical fish disease. We will replace the EGFP with ichthyophthiriasis antigen gene, or even much more genes of fish disease, and do experiments in zebra fish to test whether our DNA vaccine can work in vivo experiment or not.

Figure 1.Illness

Figure 2.Healthy

2. We will use different modes of packing to convey our DNA-protein complex to tissues that are susceptible to infection, and make preparations for oral vaccine.

3. We will extract some specific promoter of zebra fish, like histone H3 promoter and β-actin promoter. We will use these promoters to take the place of CMV promoter. This will improve the efficiency of DNA vaccine in zebra fish, and it will improve biosafety.

Figure 3.Lane 1 is marker DL5000 and lane 2 is the genome of zebrafish

1.Genome of zebrafish 2.Marker DL5000 We have extracted the genome of zebrafish and tried to get H3 promoter by PCR.

4. We will do something to modify TAT:H4 to improve it's transport efficiency.

5.We got the new sequence of holin and endolysin with the help of Peking University before the wiki freezed. We will keep testing the new self-lysis devices and try to get ideal results.

6.We also want to use quorum sensing to control the lysis. Thus we don’t need inducer to induce the lysis, which is closer to the vivo experiments.

Self-lysis device automatically

We got DNA sequences of holin and endolysin of lambda phage with the help of Peking University before the wiki freezed. We will keep testing the new self-lysis devices and trying to get expected result. And we will also use quorum sensing to control the lysis. Thus we can induce the lysis automatically instead of manually, which is closer to the requirements of vivo experiments.

Figure 4.  Self-lysis device is controled by the system of qurom sensing.

• Simplifying the RP4 plasmid
• Replace the plasmid backbone
• Conjugating various types of recipient bacterium
• Oral DNA vaccine
• Self-lysis device automatically