Position effect on secretion efficiency v1

A proposed method to evaluate secretion efficiency dependency on relative position inside the expression vector.

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.

Kloning Gen Coat Protein (CP) Carnation Mottle Virus (CarMV) pada Vektor Ekspresi [Cloning of Carnation Mottle Virus (CarMV) Coat Protein Gene into Expression Vector]

<p>Carnation mottle virus (CarMV) termasuk anggota genus Carmovirus dalam famili Tombusviridae. Virus ini banyak ditemukan menginfeksi tanaman anyelir di Jawa Barat dan menyebabkan gejala mottle. Sebagai langkah awal untuk memproduksi antiserum melalui teknik ekspresi gen CP perlu diklon pada vektor yang sesuai. Penelitian ini bertujuan mendapatkan klon CarMV yang berfungsi melalui kloning dan subkloning gen CP CarMV ke dalam vektor ekspresi yang sesuai. Penelitian dilakukan dalam beberapa tahap, yaitu ekstraksi RNA total dan amplifikasi cDNA CarMV dengan RT-PCR, menggunakan primer spesifik CarMVF dan CarMVR yang mengandung situs enzim restriksi XhoI dan BamHI, kloning dan subkloning DNA sisipan, serta konfirmasi transforman. Rekombinan gen sisipan CP CarMV dalam bakteri dikonfirmasi dengan koloni PCR. Gen CP CarMV berhasil dikloning ke dalam TA vektor pTZ57R/T dan disubkloning ke vektor ekspresi pET28a. Sekuen rekombinan CP CarMV berhasil dikonfirmasi melalui perunutan DNA. Penelitian lebih lanjut diperlukan untuk mendapatkan produksi antigen rekombinan yang melimpah pada bakteri ekspresi dan kondisi yang sesuai.</p><p><strong>Keywords</strong></p><p>Dianthus caryophillus L.; Carmovirus; Kloning; Subkloning; Bakteri ekspresi</p><p><strong>Abstract</strong></p><p>Carnation mottle virus (CarMV) is a type member of Carmovirus genus in family of Tombusvirus. The virus infects carnation plants in the centre area production of West Java and it cause mottle symptoms. The research aimed to obtain functional clone(s) of CarMV CP gene in suitable expression kloning vector. The research was carried out through several steps, namely total RNA extraction and amplification of cDNA of CP CarMV by RT-PCR using specific primer pairs CarMVF and CarMVR containing restriction enzyme sites XhoI and BamHI, respectively, TA cloning, and subcloning into expression vector pET28a and confirmation of recombinant plasmids by colony PCR. CarMV CP gen was successfully cloned into TA cloning vector pTZ57R/T and subcloned into vector pET28a, alsowere confirmed by DNA sequencing. Future experiment is necessary to be conducted to obtain abundance recombinant antigen production of CarMV CP in suitable expression condition and bacterial host.</p>

A Myo-Inositol-Inducible Expression System for Corynebacterium glutamicum and Its Application

Corynebacterium glutamicum is one of the important industrial microorganisms for production of amino acids and other value-added compounds. Most expression vectors used in C. glutamicum are based on inducible promoter (Ptac or Ptrc) activated by isopropyl-β-D-thiogalactopyranoside (IPTG). However, these vectors seem unsuitable for large-scale industrial production due to the high cost and toxicity of IPTG. Myo-inositol is an ideal inducer because of its non-toxicity and lower price. In this study, a myo-inositol-inducible expression vector pMI-4, derived from the expression vector pXMJ19, was constructed. Besides the original chloramphenicol resistance gene cat, multiple cloning sites, and rrnB terminator, the pMI-4 (6,643 bp) contains the iolRq cassette and the myo-inositol-inducible promoter PiolT1. The pMI-4 could stably replicate in the C. glutamicum host. Meanwhile, the non-myo-inositol degradation host strain C. glutamicumΔiolGΔoxiCΔoxiDΔoxiE for maintaining the pMI-4 was developed. Overexpression of hemAM and hemL using pMI-4 resulted in a significant accumulation of 5-aminolevulinic acid, indicating its potential application in metabolic engineering and industrial fermentation.

Hemin as a novel candidate for treating COVID-19 via heme oxygenase-1 induction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 (COVID-19). More than 143 million cases of COVID-19 have been reported to date, with the global death rate at 2.13%. Currently, there are no licensed therapeutics for controlling SARS-CoV-2 infection. The antiviral effects of heme oxygenase-1 (HO-1), a cytoprotective enzyme that inhibits the inflammatory response and reduces oxidative stress, have been investigated in several viral infections. To confirm whether HO-1 suppresses SARS-CoV-2 infection, we assessed the antiviral activity of hemin, an effective and safe HO-1 inducer, in SARS-CoV-2 infection. We found that treatment with hemin efficiently suppressed SARS-CoV-2 replication (selectivity index: 249.7012). Besides, the transient expression of HO-1 using an expression vector also suppressed the growth of the virus in cells. Free iron and biliverdin, which are metabolic byproducts of heme catalysis by HO-1, also suppressed the viral infection. Additionally, hemin indirectly increased the expression of interferon-stimulated proteins known to restrict SARS-CoV-2 replication. Overall, the findings suggested that HO-1, induced by hemin, effectively suppressed SARS-CoV-2 in vitro. Therefore, HO-1 could be potential therapeutic candidate for COVID-19.

Directed Expression of Tracheal Antimicrobial Peptide as a Treatment for Bovine-Associated Staphylococcus Aureus-Induced Mastitis in Mice

Bovine mastitis is perplexing the dairy industry since the initiation of intensive dairy farming, which has caused a reduction in the productivity of cows and an escalation in costs. The use of antibiotics causes a series of problems, especially the formation of bacterial antimicrobial resistance. However, there are limited antibiotic-free therapeutic strategies that can effectively relieve bacterial infection of bovine mammary glands. Hence, in this study, we constructed a mammary gland tissue-specific expression vector carrying the antimicrobial peptide of bovine-derived tracheal antimicrobial peptide (TAP) and evaluated it in both primary bovine mammary epithelial cells (pBMECs) and mice. The results showed that the vector driven by the β-lactoglobulin gene (BLG) promoter could efficiently direct the expression of TAP in pBMECs and the mammary gland tissue of mice. In addition, significant antibacterial effects were observed in both in vitro and in vivo experiments when introducing this vector to bovine-associated Staphylococcus aureus-treated pBMECs and mice, respectively. This study demonstrated that the mammary gland tissue-specific expression vector could be used to introduce antimicrobial peptide both in in vitro and in vivo and will provide a new therapeutic strategy in the treatment of bovine mastitis.

Selenoprotein T Protects Endothelial Cells against Lipopolysaccharide-Induced Activation and Apoptosis

Sepsis is an exaggerated immune response upon infection with lipopolysaccharide (LPS) as the main causative agent. LPS-induced activation and apoptosis of endothelial cells (EC) can lead to organ dysfunction and finally organ failure. We previously demonstrated that the first twenty amino acids of the Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) are sufficient to inhibit EC apoptosis. To identify genes whose regulation by LPS is affected by this N-terminal APEX1 peptide, EC were transduced with an expression vector for the APEX1 peptide or an empty control vector and treated with LPS. Following RNA deep sequencing, genes upregulated in LPS-treated EC expressing the APEX1 peptide were identified bioinformatically. Selected candidates were validated by semi-quantitative real time PCR, a promising one was Selenoprotein T (SELENOT). For functional analyses, an expression vector for SELENOT was generated. To study the effect of SELENOT expression on LPS-induced EC activation and apoptosis, the SELENOT vector was transfected in EC. Immunostaining showed that SELENOT was expressed and localized in the ER. EC transfected with the SELENOT plasmid showed no activation and reduced apoptosis induced by LPS. SELENOT as well as APEX1(1-20) can protect EC against activation and apoptosis and could provide new therapeutic approaches in the treatment of sepsis.