Summary of Biotechnological doctoral thesis: Screening and expression of a gene encoding protease inhibitor protein of microorganisms associated with the sponge Spheciospongia vesparium collected from Mientrung sea, Vietnam

Detection and screening of genes related to biosynthesis of substances with protease inhibitory activity from microorganisms associated with sponge collected from the Mien trung sea of Vietnam by metagenomics method. Then, selective gene, expression of in vitro and determination of activity of the expressed protein.
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Summary of Biotechnological doctoral thesis: Screening and expression of a gene encoding protease inhibitor protein of microorganisms associated with the sponge Spheciospongia vesparium collected from Mientrung sea, Vietnam MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ----------------------------- Tran Thi Hong SCREENING AND EXPRESSION OF A GENE ENCODINGPROTEASE INHIBITOR PROTEIN OF MICROORGANISMSASSOCIATED WITH THE SPONGE Spheciospongia vesparium COLLECTED FROM MIENTRUNG SEA, VIETNAMMajor: BiotechnologyCode: 9420201 SUMMARY OF BIOTECHNOLOGICAL DOCTORAL THESIS Ha Noi - 2020The work was completed in: Graduate university of sience andtechology- Viet Nam academy of science and technology.Science instructor 1:Assoc.Prof.PhD. Pham Viet CuongScience instructor 2:Assoc.Prof.PhD. Nguyen Thi Kim CucReviewer 1: …Reviewer 2: …Reviewer 3: …The thesis will be defended in front of the Academy Thesis EvaluationCouncil, meeting at Graduate university of sience and techology- VietNam academy of science and technology at ... hour .. ’, date ... month ...year 20........The thesis can be found at:- Library of Academy of Science and Technology-Vietnam National Library 1 PREAMBLEThe urgency of the subject Protease inhibitors (PIs), an agent that can cause protease tolose its ability to hydrolyze proteins into peptides are applied inmany fields, especially in medicine such as: antiretroviral therapy forfibrinolysis; angiotensin inhibitors in the treatment of hypertension;prevent the replication of some viruses (HIV, hepatitis C ...).Examples of some protease inhibitors are saquinavir (Brand name:Invirase) and ritonavir (Brand name: Novir) used mainly in thetreatment HIV/ AIDS. They are used as part of a multi-drug cocktailand have been shown to significantly reduce the level of HIV virus inthe blood. Therefore, the screening and detection of new proteinmolecules with specific inhibitory activity of different targetproteases, related to diseases is an interesting research direction.Sponge and its associated microorganisms are considered a source todetect and exploit new biologically active compounds for drugmanufacture, peptides with protease inhibiting activity are amongthere. However, the majority of sponge-associated microorganismsare non-cultivable, limiting their ability to exploit them. Usingmetagenomics method, with the help of high-performance genesequencing equipment and bioinformatics tools, it is possible todetect genes and gene clusters that function to encode PIs, allowingthe exploitation of PIs from non-cultivable microorganisms. Currently in Vietnam, there have been many studies onsponges, from the isolation of associated microorganisms, to theextraction of bioactive substances from this group ofmicroorganisms. However, there are no researchs using tool andnext-generation to detect and screen biologically active genes 2including protease inhibitory and expression of protease inhibitorygenes in Escherichia coli and Pichiapastoris system. Therefore, the thesis with the title: “Screening and expressionof a gene encoding protease inhibitor protein of microorganismsassociated with the sponge Spheciospongia vesparium collected fromMientrung sea, Vietnam” was carried out.Objectives of the study Detection and screening of genes related to biosynthesis ofsubstances with protease inhibitory activity from microorganismsassociated with sponge collected from the Mientrung sea of Vietnamby metagenomics method. Then, selective gene, expression of invitro and determination of activity of the expressed protein.Research content Isolation of DNA of sponge-associated microorganisms, determination of DNA concentration and purity, selection of a DNA sample for metagenomics sequencing. Analyze metagenomics data of the sequenced sample using bioinformatic tools. Assessment of the biodiversity of microorganisms associated with the sponge. From the metagenomics database, exploitation of the gene coding for substances with protease inhibitory activity. Expression of the gene encoding protease inhibitor protein in Escherichia coli and Pichia pasroris expression systems. Evaluation of protease inhibitory activity of recombinant proteins.New contributions of the thesis The DNA metagenome database of the microorganisms associated with the sponge QT2 collected at Quang Tri 3 (Mientrung) sea, Vietnam was constructed. The obtained database is scientific meaningful, contributing to the conservation of genetic resources of Vietnamese marine microorganism and has the potential for practical application. New PI-QT gene encoding PIs was obtained and successfully expressed in E. coli and P. pastoris. The anti-protease activity of the expressed protein was assessed and the obtained results showed that recombinant PI-QT protein from E. coli possed anti- trypsin, anti-ɑ-chymotrypsin activity and the protein from P. pastoris exhibited protease inhibition activity in order trypsin, ɑ- chymotrypsin and thermolysin. CHAPTER 1. OVERVIEW1.1. Sponge and sponge-associated microorganisms1.1.1. Introduction of sponge1.1.2. Sponge-associated microorganisms1.2. Overview of metagenomics1.3. Protease inhibitors (PIs)1.3.1. Summary of proteases1.3.2. Protease inhibitors and classification1.3.3. The mechanism of action of PIs1.3.4. Application of protease inhibitors1.3.5. The research situation on protease inhibitors from sponge-associated microorganisms in Vietnam and other cou ...