Cloning and Expression Analysis of bZIP Transcription Factor from Finger Millet (Eleusine coracana L.) Under Abiotic Stress Conditions

Basic leucine zipper (bZIP) transcription factors comprise one of the largest gene families in plants. They play a key role in almost every aspect of plant growth and development and also in biotic and abiotic stress tolerance. In this study, we were attempted to study characterization of bZIP, a transcription factor from a climate smart cereal finger millet (Eleusine coracana L.). Seeds of Eleusine coracana (finger millet) was purchase from local market and were grown under field conditions drought and salt stress conditions. In this study, EcbZIP gene was isolated from finger millet, cloned into DH5α cells, screened by using colony PCR and expression analysis in response to two abiotic stresses was carried out by using qRT PCR. EcbZIP coding DNA sequence and protein sequence were retrieved from NCBI Nucleotide Database and Genpept of Accession number KP033192.1 and AJP67539.1 and validated by using SMART (simple modular architecture tool) Domain Tool. Cloning and expression studies were carried out using standardized molecular biology protocol. Results depicted that EcbZIP transcription factor showed significant upregulation under both salt and drought stress conditions, indicating that it plays an important role in tolerance towards these stresses. In conclusion, expression analysis of bZIP gene from finger millet seed cultivar ML-365 showed 5-fold upregulation to salt stress to drought stress and 8-fold upregulation to salt stress. Hence, it can serve as a candidate gene for improving abiotic stress tolerance and can be helpful in enhancing the crop productivity under stress conditions.

Cloning and Expression of PirA gene of Vibrio parahaemolyticus Strain K5 Causing Acute Hepatopancreatic Necrosis Disease in Whiteleg Shrimp in E. coli Host Cell

Background: Acute hepatopancreatic necrosis disease (AHPND), is a bacterial disease of whiteleg shrimp, which has a high mortality rate (100%) and incurs economic losses. Our objective was to identify the genes which lead to cell and organ damage and investigate bioproducts to prevent and treat. Methods: Litopenaeus vannamei shrimp in Thua Thien Hue province, Vietnam were collected from an infected pond and analysed at the Institute of Biotechnology, Hue University. The PirA gene of Vibrio parahaemolyticus strain K5 was isolated and analyzed for nucleotide sequence and paired with the expression vector pQE30. The expression vector was transformed into E. coli strain M15, the PirA recombinant protein was expressed in the form of 6xHis-PirA fusion protein of about 15 kDa. PirA recombinant protein was purified and determined the PirAvp binding ratio, cloning and sequencing of PirA gene from Vibrio parahaemolyticus strain K5 causing AHPND by PCR method with specific primers and molecular weights of PirAvp and the PirAvp complex. Results: PirA gene from Vibrio parahaemolyticus strain K5 was cloned into pGEM-T easy vector (Promega, USA) and screened E. coli TOP10 colonies containing pGEM T easy/PirA recombinant plasmid on LB agar/ampicillin/IPTG/X-Gal medium. PCR showing a band of about 347 bp, matching the size of PirA gene and two nucleotide sequences (BamHI and HindIII). The results showed that PirA gene has a length of 336 bp and similar to PirA gene on GenBank (Code: KU556825.1). The results of protein extracted from E. coli M15 recombinant cells and 6xHis-PirA target protein was collected in elution fractions from EF2 to EF6, showed that the concentration of 6xHis-PirA protein and EF3 elution fraction collected a highest protein concentration (1,586.54 µg/ml). Conclusions: The purified PirA recombinant protein will provide materials for development research to create biological products to prevent and treat AHPND.

Identification of mercury‐resistant Streptomyces isolated from Cyperus rotundus L. rhizosphere and molecular cloning of mercury (II) reductase gene

Streptomyces is one of mercury‐resistant bacteria which can convert Hg2+ into nontoxic Hg0 . This study aimed to identify mercury‐resistant Streptomyces present in the Cyperus rotundus rhizosphere from artisanal small‐scale gold mining (ASGM) area and clone merA gene to the cloning and expression vectors. Molecular identification was conducted using 16s rRNA gene with the maximum likelihood algorithms. Results revealed that the AS1 and AS2 strains were a group of Streptomyces ardesiacus and the BR28 strain was closed to Brevibacillus agri. The AS2 merA gene was cloned to pMD20 cloning vectors, pGEX‐5x‐1 and pET‐28c expression vectors. The transformation was successfully performed in BL21 and DH5α competent cells. The full length of the merA gene was confirmed to be 1,425 bp. This study is the first research on identifying mercury‐resistant Streptomyces and cloning the full‐length merA gene in Indonesia.

Cloning and expression of the bacteriophage-derived endolysin against Aeromonas hydrophila

Hemorrhagic septicemia disease in striped catfish is caused by Aeromonas hydrophila bacterium. Antibiotics are commonly used to treat this disease, however, due to antibiotic resistance in A. hydrophila, it is necessary to have an alternative antibacterial agent to antibiotics. Endolysins are bacteriophage-encoded peptidoglycan hydrolases that are synthesized at the end of the lytic phage replication cycle, they lyse the host bacterial cell wall and release new bacteriophage virions. In this study, an endolysin (cell wall hydrolase) derived from A. hydrophila phage PVN02 was artificially synthesized, cloned into pET28a(+) and successfully expressed in E. coli BL21 (DE3). The recombinant endolysin, cell wall hydrolase strongly exhibited antimicrobial activity against A. hydrophila with a reduction of 3-log CFU/ml of A. hydrophila after 30 minutes of mixing and further 30 minutes of incubation, the bacterial cells were lysed completely. It should be emphasized that the lytic activity by the recombinant endolysin to A. hydrophila bacteria did not require a pretreatment with an outer-membrane permeabilizer. The results of our study showed a potential of use this recombinant endolysin as a novel antibacterial agent to replace antibiotics in the treatment of hemorrhagic septicemia diseases in striped catfish.

64 A cloning and expression system of the neoantigen-specific TCRs from tumor-infiltrating lymphocytes by single-cell sequencing of paired TCRα and TCRβ chains

BackgroundT-cells that target tumor neoantigens arising from cancer mutations are the primary mediators of cancer immunotherapies. Identifying neoantigens and T-cells that recognize them is essential for T-cell-based immunotherapy. However, neoantigen-reactive Tumor-infiltrating lymphocytes (TILs) are highly differentiated or exhausted with a limited proliferative capacity; it is challenging to expand them for a sufficient number to probe their specificity. Therefore, we developed a novel cloning and expression system to examine TCRs discovered by single-cell sequencing of TILs for their neoantigen-specificity.MethodsTILs of lung cancer and sarcoma were analyzed. Surgically removed tumors were divided into several pieces. They were enzymatically digested to prepare fresh tumor digest (FTD) and cryopreserved. They were used to generate TIL cultures and perform WES and RNA-Seq to identify tumor-specific mutations. MHCflurry was used to predict the binding affinity of potential epitopes arising from these mutations to HLA class I. Peptides that were predicted to bind to patients‘ own MHC class I molecules strongly were then synthesized. Single TILs isolated with the ICELL8® cx system (Takara Bio) were dispensed into a nanowell TCR chip containing preprinted barcodes. Barcoded cDNAs were PCR-amplified in-chip, pooled off-chip, and used as a template in the TCR-specific PCR or for the whole transcriptome library generation of 5’ ends of all transcripts. Based on single-cell transcriptome data and TCR profiles of TILs, we predict and prioritize neoantigen-specific TCRs and cloned them into siTCR® retrovirus vectors. These TCRs were transduced into SUP-T1-based reporter cells in which ZsGreen fluorescent protein expression is controlled by AP-1 and NFAT binding sites. TCR-expressing reporter cells were cocultured with patient autologous APCs pulsed with a pool of candidate neoantigen peptides. ZsGreen expression indicates that TCRs match their cognate neoantigens.ResultsIn a lung cancer patient, we set up 18 TIL cultures and obtained 12 TILs. TILs were cocultured with FTD; IFN-γ production was measured by ELISA to evaluate their reactivity to the autologous tumor. NGS identified 197 somatic mutations, 4 fusion genes, and 8 highly expressed cancer-testis antigens. Among them, 339 candidate peptides were synthesized and screened. In addition, we cloned 3 pairs of TCRαβ chains from most expanded TIL cultures and 4 TCRs from ex vivo TILs with exhausted phenotype. Two reporter cells that express TCRs from exhausted TILs responded to the same neoantigen peptide.ConclusionsGenerating TCR expressing cell lines facilitated the identifying neoantigens and their cognate TCR sequences from patients.Ethics ApprovalG3545