1. Will the exogenous DNA fuse into genome?

At present, the FDA has authorized malaria, HIV, Ebola and cancer's DNA vaccine to have clinical test. In 2005, West Nilelnnovator DNA vaccine and Salmon infectious hemorrhagic necrosis virus DNA vaccine APEX-IHN had come onto market.

We had searched a lot of papers and data, and there is no evidence showing that exogenous DNA can fuse into the genome . From the paper, we know that Ledwith had injected mice' muscle by DNA vaccine. After 6 weeks, the plasmid residues are 1000-4000 copies/ug in the injection area. After 6 month, it became 200-800 copies/ug. And the probability of fusion is <=1-8 per 150000 cells, that means the possibility of DNA fusion in every cell is less than 6.7x10-6, however the possibility in normal cell is higher 3 order of magnitudes. So, researchers believe that the fusion caused by exogenous DNA injection can be ignored.

2. Is the TAT-H4 fusion protein safety?

At present, TAT is a customary way to mediate cellular delivery, and recombinant plasmids can be introduced into living cells. A method using macro-branched TAT has been proposed for plasmid DNA delivery into various cell lines and showed significant transfection capabilities. Some iGEM teams also use this in their design and it has been proved safe in mediated cellular delivery. It's a part of HIV-1 transactivating protein called protein transduction domain. It' s natural function is only helping the transactivating protein transfect into cells, because it is just an non-core part of the transactivating protein so it won't cause harm for human or other organisms. In many researches, the TAT-PDT was used to transport medicinal protein to kill cancer cells, and there is no evidence showing the TAT-PDT is unsafe. As Histone H4 protein, many studies explored the possibility of using non-viral, plant-based gene sequences to create strong and constitutive expression vectors. Wheat H4 histone comes from plant and many labs also use it in DNA vaccine, so we don’t need to worry too much about safety.

3. Will the protein cause immune response in fish?

The report about fish immunity is concerned with DNA vaccine, their experiment to answer the question of specificity would be to vaccinate fish with a DNA vaccine for target gene, and the vector is only a way to mediate the target gene, such as in mammals, IFN released from virus-infected cells bind to specific receptors on the plasma membrane in an autocrine as well as in paracrine fashion, up regulating the expression of over 20 IFN-regulated proteins that can directly or indirectly interfere with viral replication. Researchers detect the antibodies in vaccinated fish and, the results are aimed at different genes, so we don’t need to worry about this new approach, and this will be a new and efficient pattern for vectors.

4.How to resolve the spread of resistance plasmid RP4?

Throughout the project, the main safety threat would be the transfer of plasmid carrying genes for ampicillin, kanamycin and tetracycline resistance, which result from conjugation between bacteria. However, we have come up with tactics aiming at the issue. The original plasmid RP4 contains the sequence of tra1 and tra2 that contribute to Mpf (mating pair formation) and the oriT (the transfer origin) site which is the start of conjugation. Under certain conditions, plasmid RP4 with tra1, tra2 and oriT site could conjugate between cells.

In order to prevent plasmid RP4 from transferring in other cells, we deactivate the oriT site, thus there’s no designated transfer origin could be recognized. Then only the Mpf could be formed, but the transferring function would lost. In the meantime, we construct another mini plasmid which merely carries oriT but no tra1 and tra2, especially no resistance genes. Subsequently, with the help of plasmid RP4 of which the oriT site is deactivated, under the Mpf is formed, the mini plasmid could transfer through the cell envelopes of the donor and the recipient cell. Therefore, we prevent the resistance plasmid RP4 transferring and make the mini plasmid carrying functional genes enter the recipient cell to perform the specific task, and then we solve the safety threat effectively.

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