Cloning and expression of \(\textit{ pigI}\) gene in \(\textit{Escherichia coli}\) BL21 (DE3)

Prodigiosin (Pg), a secondary metabolite with anticancer and antimicrobial activities, can be produced in Serratia marcescens bacteria through the condensation reaction of 4-methoxy-2, 2’-bipyrrole-5-carboxyaldehyde (MBC) and 2-methyl-3-amylpyrrole (MAP). Among these, the MBC synthetic pathway is started by the conversion of L-proline to L-proline-AMP before this complex is covalently attached to PigG. This reaction is catalyzed by an L-prolyl-AMP ligase named PigI. Therefore, PigI protein plays an important role in the prodigiosin biosynthetic pathway. However, studies related to PigI protein have not been carried out in Vietnam yet. In this work, the pigI gene was cloned and expressed in Escherichia coli DH10B and BL21 (DE3), respectively. Sequence alignment results revealed that the obtained pigI gene is 99.7% identical to the four strains, CP027798, CP027796, CP021984 and CP003959. This recombinant vector pJET1.2/pigI was used to reamplify pigI, and the acquired amplicon was inserted into pET22b vector at the site of HindIII and XhoI. The clone E. coli BL21 (DE3) containing the recombinant vector pET22b/pigI was expressed in an auto-induced medium. The presence of PigI protein in the lysate was identified due to a 53 kDa band through Western Blot analysis using an anti-his-tag antibody. The results of our study provide a potential method for producing prodigiosin from recombinant protein in Vietnam.

SINE Insertion in 5’ Flanking of IGFBP3 May Associated With Gene Expression and Phenotypic Variations

Background: Insulin-like growth factor binding proteins (IGFBPs), specifically binding to IGF1 and IGF2, play an important role in regulating physiological functions of insulin-like growth factors (IGFs). IGFBPs have been considered important candidate genes for economic traits due to their involvement in physiological processes related to growth and development. However, most of the current studies on genetic markers of IGFBPs have focused on SNPs, and large fragment insertion mutations such as retrotransposons have rarely been considered. In this paper, we screened the porcine IGFBP genes (IGFBP1-8) for retrotransposon insertion polymorphisms (RIPs) using bioinformatics prediction combined with the PCR-based amplification. Furthermore, for two linked RIPs their population distribution and impact on promoter activity and phenotype were further evaluated.Results: Screening of IGFBPs identified RIPs in IGFBP1-5 and IGFBP7. In total twelve predicted RIPs were confirmed by PCR. These RIPs were detected in different breeds with an uneven distribution among them. By linkage genetic analysis and PCR verification, IGFBP3-1-RIP and IGFBP3-2-RIP are completely linked, showing only three genotypes, SINE+/+/LINE-/-, SINE-/-/LINE+/+ and SINE+/-/LINE-/+. The age of 100 kg body weight and longissimus muscle thickness of Large white individuals of SINE+/+/LINE-/-genotype were significantly (P<0.05) higher than those of SINE+/-/LINE+/- genotype and SINE-/-/LINE+/+ genotype. However, the longissimus muscle thickness and corrected backfat thickness of SINE+/+/LINE-/- individuals were significantly (P<0.05) thinner than those of SINE+/+/LINE-/- genotype. The expression of the IGFBP3 gene in liver, leg muscles and backfat of 30-day Sujiang piglets with SINE+/+/LINE-/- genotype were significantly higher (P<0.05) than those with SINE-/-/LINE+/+ genotype by the real-time quantitative polymerase chain reaction (qPCR). Further study was conducted to confirm the effect of SINE and LINE insertion on the promoter activity of IGFBP3. First, the core promoter region of the IGFBP3 gene was identified locating within 482bp upstream of ATG by using the dual-luciferase activity assay. Then SINE and LINE were combined with 482bp fragment to construct a recombinant vector respectively based on the PGL3-Promoter-Enhancer. The recombinant vector was transfected into C2C12, 3T3-L1, and Hela cells. The detection of the dual-luciferase reporter gene revealed that only SINE insertion was significantly increased (P<0.05) promoter activity of the IGFBP3 gene, indicating that the SINE may act as an enhancer to regulate the promoter activity of the IGFBP3 gene. Conclusions: Overall, this study identified 12 RIPs in IGFBP gene clusters, which provided useful markers for genetic analysis of pig populations. Furthermore, based on the dual-luciferase activity assay in cells and association analysis, the linked genetic variations generated by SINE and LINE insertions in 5’ flanking of IGFBP3 may associated with variations of gene expression and phenotype.

Cloning and expression of pigC gene in Escherichia coli

Prodigiosin (Pg), which is particularly of interest because of anticancer and antimicrobial activities, can be produced through the PigC-catalyzed condensation reaction of 4-methoxy-2, 2’-bipyrrole-5-carboxyaldehyde (MBC) and 2-methyl-3-amylpyrrole (MAP). Therefore, the PigC protein plays an important role in prodigiosin biosynthetic pathway. However, studies related to PigC protein have not been carried out in Vietnam yet. In this work, the pigC gene was cloned and expressed in Escherichia coli DH10B and BL21 (DE3), respectively. Using PCR and universal primers, we amplified a fragment of 3 kb covering entire coding region of the pigC gene from Serratia sp. strain M5. The pigC gene was inserted into pJET1.2 vector, and then transformed into E. coli DH10B. The sequence of a recombinant vector pJET1.2/pigC was evaluated by using whole colony PCR amplification. Sequence alignment results revealed that the obtained pigC gene possesses 71.5% and 75.4% of nucleotide identity in comparison with two strains, Serratia 39006 and Serratia sp. AS9 published in GenBank with their respective accession numbers of AJ833001 and CP002773. The recombinant vector pJET1.2/pigC was used to reamplify pigC, and the acquired amplicon was inserted into pET22b vector at the site of HindIII and XhoI. The clone E. coli BL21 (DE3) containing recombinant vector pET22b/pigC was expressed in the auto-induced medium. The presence of PigC protein in the lysate was identified as a 100 kDa band through Western Blot analysis using anti his-tag antibody. Afterward, the PigC protein was purified by Ni-NTA column, and its expression level was quantified through SDS-PAGE analysis. The results of our study provide a potential material for producing prodigiosin from recombinant protein in Vietnam.

Evaluation of the Immune Response Against Helicobacter pylori in Infused BALB/c Mice by pcDNA3.1(+)-ureA

Background: The purpose of the present study was to produce a pcDNA3.1(+)-ureA recombinant vector and evaluate the capacity of this vector to stimulate the immune response against H. pylori infection in infused BALB/c mice.&nbsp; &nbsp; Materials and methods: The pcDNA3.1(+)-ureA construct was prepared and transformed into E. coli, successfully. The animals we used in the study were allotted into three groups for infusion of 1) recombinant plasmid, 2) pcDNA3.1(+)-ureA + nanoparticles, and 3) pcDNA3.1(+). Blood and tissue specimens from each group of mice were collected at days 15, 30, and 45 after the last infusion and the expression levels of cytokines such as TGF-&beta;1, IL-4, and IFN&gamma; genes comparing to GAPDH as well as the expression of ureA in the mice&rsquo;s thigh muscle were evaluated.&nbsp; &nbsp; Results: The genes expression analysis showed that the IL4 expression significantly decreased (p<0.001) but IFN&gamma; and TGF-&beta;1 expression increased in the blood of infused mice (p<0.001). Also, the urea expression level in pcDNA3.1(+)-urea and pcDNA3.1(+)-ureA+ nanoparticle 15, 30, and 45 days after the last infusion was significantly different (p<0.001) and its expressions at days 15 and 30 were significantly different (p<0.001), but 45 days after the last infusion it was not significantly different (p>0.05).&nbsp; &nbsp; Conclusion: The pcDNA3.1(+)-ureA recombinant vector with or without chitosan nanoparticles can stimulate the immune response in animal models against H. pylori infection. Also, after combining the recombinant vector with nanoparticles we observed a better immune response was observed. In future studies this recombinant construct can be used as a biomarker and therapeutic approaches in eukaryotic systems.