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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 ENCODING PROTEASE INHIBITOR PROTEIN OF MICROORGANISMS ASSOCIATED WITH THE SPONGE Spheciospongia vesparium COLLECTED FROM MIENTRUNG SEA, VIETNAM Major: Biotechnology Code: 9420201 SUMMARY OF BIOTECHNOLOGICAL DOCTORAL THESIS Ha Noi - 2020 The work was completed in: Graduate university of sience and techology- Viet Nam academy of science and technology. Science instructor 1:Assoc.Prof.PhD. Pham Viet Cuong Science instructor 2:Assoc.Prof.PhD. Nguyen Thi Kim Cuc Reviewer 1: … Reviewer 2: … Reviewer 3: … The thesis will be defended in front of the Academy Thesis Evaluation Council, meeting at Graduate university of sience and techology- Viet Nam 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 PREAMBLE The urgency of the subject Protease inhibitors (PIs), an agent that can cause protease to lose its ability to hydrolyze proteins into peptides are applied in many fields, especially in medicine such as: antiretroviral therapy for fibrinolysis; 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 the treatment HIV/ AIDS. They are used as part of a multi-drug cocktail and have been shown to significantly reduce the level of HIV virus in the blood. Therefore, the screening and detection of new protein molecules with specific inhibitory activity of different target proteases, related to diseases is an interesting research direction. Sponge and its associated microorganisms are considered a source to detect and exploit new biologically active compounds for drug manufacture, peptides with protease inhibiting activity are among there. However, the majority of sponge-associated microorganisms are non-cultivable, limiting their ability to exploit them. Using metagenomics method, with the help of high-performance gene sequencing equipment and bioinformatics tools, it is possible to detect genes and gene clusters that function to encode PIs, allowing the exploitation of PIs from non-cultivable microorganisms. Currently in Vietnam, there have been many studies on sponges, from the isolation of associated microorganisms, to the extraction of bioactive substances from this group of microorganisms. However, there are no researchs using tool and next-generation to detect and screen biologically active genes 2 including protease inhibitory and expression of protease inhibitory genes in Escherichia coli and Pichiapastoris system. Therefore, the thesis with the title: “Screening and expression of a gene encoding protease inhibitor protein of microorganisms associated with the sponge Spheciospongia vesparium collected from Mientrung sea, Vietnam” was carried out. Objectives of the study Detection and screening of genes related to biosynthesis of substances with protease inhibitory activity from microorganisms associated with sponge collected from the Mientrung sea of Vietnam by metagenomics method. Then, selective gene, expression of in vitro 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 antitrypsin, anti-ɑ-chymotrypsin activity and the protein from P. pastoris exhibited protease inhibition activity in order trypsin, ɑchymotrypsin and thermolysin. CHAPTER 1. OVERVIEW 1.1. Sponge and sponge-associated microorganisms 1.1.1. Introduction of sponge 1.1.2. Sponge-associated microorganisms 1.2. Overview of metagenomics 1.3. Protease inhibitors (PIs) 1.3.1. Summary of proteases 1.3.2. Protease inhibitors and classification 1.3.3. The mechanism of action of PIs 1.3.4. Application of protease inhibitors 1.3.5. The research situation on protease inhibitors from spongeassociated microorganisms in Vietnam and other countries 1.4. Expression system Escherichia coli and Pichia pastoris CHAPTER II: MATERIALS AND METHODS 2.1. Research materials 2.1.1. Materials 4 The sponge were collected from Quang Tri sea, Vietnam and some other materials used in the study. 2.1.2. Research subjects Sponge-associated microorganisms 2.1.3. Chemistry The chemicals used in the research were mainly purchased from reputable brands such as Merck (Germany), Sigma (USA), Himedia (India). 2.1.4. Devices Machines and equipments of Nghia Do Molecular Biology Center, Mientrung Institute for Scientific Research, Vietnam Academy of Science and Technology and some equipmentsof the Institute of Marine Biochemistry, Vietnam Academy of Science and Technology. 2.1.5. Solution and medium of use Medium used in culture and gene expression in E. coli: Luria-Bertani (LB). Medium used in transformation and expression of foreign genes in P. pastoris yeast (according to the instructions of PichiaExpression Kit). The solution used in DNA electrophoresis: TAE 1X, loading dye 6X, ethidium bromide (EtBr) 0.5 µg/mL. SDS-PAGE electrophoresis solution: Bis-acrylamide 30 %; TrisHCl buffer 1.5 M pH 8.8; TrisHCl 1 M pH buffer 6.8; TrisHCl buffer 0.5 M pH 6.8; SDS 10 %; APS 10 %; TEMED ... Methods 2.2.1. Method of isolating DNA metagenome of sponge-associated microorganisms 5 Isolation of total DNA of sponge-associated microorganisms by method of Abe et al. (2014) with some minor improvements to suit the conditions of Vietnam. 2.2.2. Sponge identification by molecular biology (Medlin et al., 1988) 2.2.3. Analysis of metagenomics data Sequencing DNA metagenome by Illumina HiSeq 2500 system (BaseClear - Netherlands). The collected data were analyzed by bioinformatic tools. 2.2.4. Synthesis of PIs gene from database metagenomics and design plasmid carrying PIs gene The PI-QT gene was screened from the metagenomic QT2 database, synthesized and cloned into the pUC57 vector, with the restriction enzymes EcoRI and NotI at both ends (GenScipt, USA). Next, the vector pUC57 (Thermo Fisher Scientifc, USA) containing the gene PI-QT and expression vector pET-32a(+) (Invitrogen, USA) were digested with restriction enzymes EcoRI and NotI (Fermentas, Lithuania). The gene PI-QT was then purifed and inserted into expression vector pET-32a(+) (expression in E. coli) and pPIC9 (expression in P. pastoris) using enzyme T4 ligase (Thermo, USA). 2.2.5. Methods used for expression and recovery of recombinant PIs in the E. coli BL21(DE3) expression system Methods of transformation, expression and optimization of gene expression in E. coli cells according to Do ThiHuyenet al., 2008; Nguyen ThiQuyet al., 2013. Western blot analysis (according to the instructions of Invitrogen, USA). Purification of recombinant PIs protein: Purification of the recombinant protein PI-QT was performed by chromatography on Ni-NTA afinity chromatography column according to the Ni-NTA 6 Purification System (Novex by life technologies) with some minor modifications. Remove the TRx-His-tag peptide from the recombinant protein (Novagen's instructions). Identification and analysis of proteins by proteomics/ bioinformatic tools Protein Biochemistry Department, Institute of Biotechnology). 2.2.6. Methods used to for expression and recovery of recombinant PIs in the Pichiapastoris expression system According to the instructions of the Pichia expression kit (Invitrogen). 2.2.7. Protease inhibitory assay (Sapna., 2013; Jiang et al., 2011) 2.2.8.Characterization of protease inhibitor PI-QT protein (Sapna., 2013). 2.2.9. Statistical Analysis The assays were performed in triplicate and expressed as the mean ± standard error of the mean (SEM). The statistical analysis was performed by t-test and one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests using the SPSS v.22 (SPSS Inc, Chicago, IL, USA). The results were considered to be signifcant at P < 0.05. CHAPTER III: RESULTS AND DISCUSSION 3.1. Collect of sponge samples Use SCUBA method, 8 samples of sponge were collected at Quang Tri sea. Sponges were collected at the coordinates of 107°07'06.0"E; 17°04'50.2"N. Samples were stored in 30 % glycerol, preserved in ice, transported to the laboratory and DNA extraction of sponge-associated microorganismswas carried out. 3.2. DNA metagenome extraction results 7 3.2.1. Extraction of DNA metagenome of microorganism associated withsponge collected at Quang Tri sea Total DNA of sponge-associated microorganism was extracted from 8 sponge specimens collected at the Quang Tri Sea, Vietnam. The results showed that the amount and quality of DNA extracted was different for each sample. The most isolated DNA was from QT2 sample and it had highest purity (Figure 3.1, Table 3.1). QT9 QT8 QT7 QT6 QT5 QT4 QT2 QT3 M Figure 3.1. Electrophoresis of isolated DNA from QT2 spongeassociated microorganisms (M: Marker 100 bp of Thermo) Table 3.1. The quality of total DNA extracted from spongescollected Serial 1 2 3 4 Sample QT2 QT3 QT4 QT5 in Quang Tri sea DNA (ng/µL) A260/A230 202,5 2,0 165,1 1,8 160,9 1,83 178,7 1,82 A260/A280 1,80 1,71 1,54 1,32 8 5 6 7 8 QT6 QT7 QT8 QT9 39,8 73,5 125,8 36,5 1,9 1,8 1,77 1,78 1,62 1,72 1,41 1,70 The total DNA of sponge-associated microorganisms QT2 was selected (DNA concentration: 202,5ng/µl, purity A260 / A280 = 1.80) for metagenomic sequencing. 3.1.2. Identify the QT2 sponge by molecular technique The nearlyfull-length 18S rRNA gene fragment of the QT2 sponge sample were amplified successfully (Figure 3.2b). The obtained sequence was compared with other sequences of 18S rRNA using the BLAST (Basic Local Alignment Search Tool) on NCBI (National Center for Biotechnology Information). The result that the 18S rRNA gene sequence of QT2 sponge was similar to 99,9 % with that of Spheciospongia vesparium sponges with code AY734440. Therefore, the QT2 sample was named Spheciospongia vesparium QT2. 3.3. Establishment of a DNA metagenome database of microorganismsassociated with Spheciospongiavesparium QT2 by bioinformatics After sequencing the entire shortgunmetagenome of the QT2 sample (Base Clear, Netherlands) and purifying the data using Trimmomatic software, over 44 million paired reads were collected. The purified data were used to assemble de novo metagenome using SPAdes software. The total size of assembly is approximately 418 Mb including 102,236 contigs. The longest contig is over 855 kb, the smallest contigs is 1,000 bp, the average length is 4,089 bp. Nearly 90 % of the sequences can be mapped back to the assembly genome. The obtained results showed that the contigs mainly distributed