Vanessa Galli

Efficient site-directed mutagenesis using an overlap extension-PCR method for expressing Mycoplasma hyopneumoniae genes in Escherichia coli.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and results in significant economic losses in swine production worldwide. Vaccination is considered to be the most cost-effective strategy for control and prevention of this disease. However, the development of new recombinant subunit vaccines is often hampered by the unusual codon usage of this bacterium. To express M. hyopneumoniae proteins in heterologous systems such as Escherichia coli, the TGA codons that encode tryptophan in M. hyopneumoniae genes need to be replaced with the TGG codon. In this study we employed a modified overlap extension-PCR method for site-directed mutagenesis of selected TGA codons. Primers carrying the appropriate TGA to TGG mutation were employed in a two-step PCR amplification. The mutated PCR products were subsequently cloned into E. coli expression vectors. Using this method, we obtained 14 M. hyopneumoniae genes with up to three TGA to TGG substitutions per gene. Expression of the 10 mutated genes in E. coli was achieved. The method was rapid, simple and highly efficient in introducing the desired mutations in the A+T rich M. hyopneumoniae genes. In conclusion, this modified overlap extension-PCR method is suitable for large-scale site-directed mutagenesis of M. hyopneumoniae genes for heterologous expression.

Carotenoid Biosynthetic and Catabolic Pathways: Gene Expression and Carotenoid Content in Grains of Maize Landraces

Plant carotenoids have been implicated in preventing several age-related diseases, and they also provide vitamin A precursors; therefore, increasing the content of carotenoids in maize grains is of great interest. It is not well understood, however, how the carotenoid biosynthetic pathway is regulated. Fortunately, the maize germplasm exhibits a high degree of genetic diversity that can be exploited for this purpose. Here, the accumulation of carotenoids and the expression of genes from carotenoid metabolic and catabolic pathways were investigated in several maize landraces. The carotenoid content in grains varied from 10.03, in the white variety MC5, to 61.50 μg·g−1, in the yellow-to-orange variety MC3, and the major carotenoids detected were lutein and zeaxanthin. PSY1 (phythoene synthase) expression showed a positive correlation with the total carotenoid content. Additionally, the PSY1 and HYD3 (ferredoxin-dependent di-iron monooxygenase) expression levels were positively correlated with β-cryptoxanthin and zeaxanthin, while CYP97C (cytochrome P450-type monooxygenase) expression did not correlate with any of the carotenoids. In contrast, ZmCCD1 (carotenoid dioxygenase) was more highly expressed at the beginning of grain development, as well as in the white variety, and its expression was inversely correlated with the accumulation of several carotenoids, suggesting that CCD1 is also an important enzyme to be considered when attempting to improve the carotenoid content in maize. The MC27 and MC1 varieties showed the highest HYD3/CYP97C ratios, suggesting that they are promising candidates for increasing the zeaxanthin content; in contrast, MC14 and MC7 showed low HYD3/CYP97C, suggesting that they may be useful in biofortification efforts aimed at promoting the accumulation of provitamin A. The results of this study demonstrate the use of maize germplasm to provide insight into the regulation of genes involved in the carotenoid pathway, which would thus better enable us to select promising varieties for biofortification efforts.

Cloning and purification of recombinant proteins of Mycoplasma hyopneumoniae expressed in Escherichia coli.

Mycoplasma hyopneumoniae, the etiological agent of swine enzootic pneumonia, is an important pathogen in the swine industry worldwide. Vaccination is the most cost-effective strategy for controlling and prevention of this disease. However, investigations on pathogenicity mechanisms as well as current serological detection methods and the development of new recombinant subunit vaccines are hampered by the lack of known and well characterized species-specific M. hyopneumoniae antigens. In this work, 54 predicted genes encoding proteins with potential to be used as subunit vaccine or antigens in diagnostic tests were selected, amplified by PCR and cloned into Escherichia coli expression vectors. Recombinant protein expression, solubility and yields were analyzed. The majority of the recombinant proteins were expressed in inclusion bodies. After solubilization with urea or N-lauroyl sarcosine, recombinant proteins were purified by Ni(2+) affinity chromatography. This approach allowed purification of thirty recombinant M. hyopneumoniae proteins which will be evaluated as vaccine candidates and/or as antigens to be used in diagnostic tests.

Immunisation of mice with Mycoplasma hyopneumoniae antigens P37, P42, P46 and P95 delivered as recombinant subunit or DNA vaccines

Porcine enzootic pneumonia (PEP), which is caused by the fastidious bacterium Mycoplasma hyopneumoniae, is one of the most economically important diseases in the pig industry worldwide. Commercial bacterins provide only partial protection; therefore, the development of more efficient vaccines against PEP is necessary. In this study, the cellular and humoral immune responses elicited by DNA and recombinant subunit vaccines based on the P37, P42, P46 and P95 antigens of M. hyopneumoniae were evaluated after the intramuscular inoculation of BALB/c mice. The expression of the cytokines INFγ, TNFα and IL1 was evaluated by real-time RT-PCR in splenocytes from vaccinated mice. All antigens delivered as subunit vaccines, especially P42 and P95, and the pcDNA3/P46 DNA vaccine were able to elicit strong immune responses. These vaccines induced cellular immune responses and the production of antibodies able to react with native M. hyopneumoniae proteins. Because both cellular and humoral immune responses were induced, P42 and P95 are promising candidates for a recombinant subunit vaccine and P46 is a promising candidate for a DNA vaccine against PEP.

Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, is highly prevalent worldwide and causes major economic losses to the pig industry. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. The aim of this study was to evaluate 34 recombinant proteins of M. hyopneumoniae expressed in Escherichia coli. Antigenic and immunogenic properties of these proteins were analyzed. For this, the proteins were tested against hyperimmune and convalescent pig sera through ELISA and Western blot. Immunogenicity of the recombinant proteins was evaluated in BALB/c mice following intramuscular inoculation. Most antigens were able to induce a strong immune response and sera from inoculated mice were able to recognize native proteins by cell ELISA and Western blot. Several recombinant proteins were specifically recognized by convalescent pig sera, indicating they are expressed during infection. These data may help to develop more efficacious vaccines against M. hyopneumoniae.

Production and characterization of recombinant transmembrane proteins from Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), a chronic respiratory disease which causes significant economic losses to the swine industry worldwide. More efficient strategies for controlling this disease are necessary. In this study, we cloned17 genes coding for transmembrane proteins from M. hyopneumoniae, among which six were successfully expressed in Escherichia coli and had their immunogenic and antigenic properties evaluated. All proteins were immunogenic in mice and sera from naturally infected pigs reacted with the recombinant proteins, suggesting that they are expressed during infection. These antigens may contribute for the development of new recombinant vaccines and diagnostic tests against EP.

Local and systemic immune responses induced by a recombinant chimeric protein containing Mycoplasma hyopneumoniae antigens fused to the B subunit of Escherichia coli heat-labile enterotoxin LTB.

A multi-antigen chimera composed of three antigens of Mycoplasma hyopneumoniae (R1, P42, and NrdF) and the mucosal adjuvant Escherichia coli heat-labile enterotoxin B subunit (LTB) was constructed, and its antigenic and immunogenic properties were evaluated in mice and pigs. In addition, we compared the effect of the fusion and co-administration of these proteins in mice. Antibodies against each subunit recognized the chimeric protein. Intranasal and intramuscular immunization of mice with the chimeric protein significantly increased IgG and IgA levels in the serum and tracheobronchial lavages, respectively, against some of the antigens present in the chimeric. Swine immunized with the chimeric protein developed an immune response against all M. hyopneumoniae antigens present in the fusion with a statistically significant difference (P<0.05). The adjuvant rLTB enhanced the immune response in both fused and co-administered antigens; however, better results were obtained with the chimeric protein. This multi-antigen is a promising vaccine candidate that may help control M. hyopneumoniae infection.

Recombinant Secreted Antigens from Mycoplasma hyopneumoniae Delivered as a Cocktail Vaccine Enhance the Immune Response of Mice

ABSTRACT Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), which is a respiratory disease responsible for huge economic losses in the pig industry worldwide. The commercially available vaccines provide only partial protection and are expensive. Thus, the development of alternatives for the prophylaxis of EP is critical for improving pig health. The use of multiple antigens in the same immunization may represent a promising alternative. In the present study, seven secreted proteins of M. hyopneumoniae were cloned, expressed in Escherichia coli, and evaluated for antigenicity using serum from naturally and experimentally infected pigs. In addition, the immunogenicity of the seven recombinant proteins delivered individually or in protein cocktail vaccines was evaluated in mice. In Western blot assays and enzyme-linked immunosorbent assays, most of the recombinant proteins evaluated were recognized by convalescent-phase serum from the animals, indicating that they are expressed during the infectious process. The recombinant proteins were also immunogenic, and most induced a mixed IgG1/IgG2a humoral immune response. The use of these proteins in a cocktail vaccine formulation enhanced the immune response compared to their use as antigens delivered individually, providing evidence of the efficacy of the multiple-antigen administration strategy for the induction of an immune response against M. hyopneumoniae.

Extraction and Quantification of Abscisic Acid and Derivatives in Strawberry by LC-MS

Phytohormones are important plant components that are involved in a signaling cascade in plant development. In strawberry, the influence of abscisic acid (ABA) and its different forms [phaseic acid (PA), dihydrophaseic acid (DPA), and ABA glucose ester (ABA-GE)] on the process of fruit maturation is not yet completely understood. Quantification of phytohormones is currently performed by liquid chromatography coupled to mass spectrometry (LC-MS) due to its sensitivity and specificity. However, the sample matrix and the extraction procedure will influence the analysis. Thus, this study aimed to optimize a simple extraction method and validate the LC-MS quantification of ABA as well as the identification and quantification of ABA derivatives (PA, DPA, and ABA-GE) in strawberry fruit. Hormone extraction was performed using either methanol (80% v/v—S1) or acetone:water:acetic acid (80:19:1 v/v—S2) solutions with or without the use of sonication. The most efficient extraction was obtained using S1 without sonication, and LC-MS validation parameters for ABA were within acceptable scores.

An improved method for characterization of the mutation associated to porcine stress syndrome by PCR amplification followed by restriction analysis

A mutation in the gene coding for the ryanodine receptor 1 (RYR1), also known as halothane (hal) gene or swine stress gene, is associated to the porcine stress syndrome (PSS). Detection of the mutation is normally accomplished by PCR amplification of an 81bp fragment of the hal gene, followed by digestion with the HhaI restriction endonuclease. Wild-type allele (N) is cut in two fragments, whereas the mutant allele (n) is not digested by the restriction enzyme. Electrophoresis of the digested DNA on agarose gel and ethidium bromide staining allows the reading of the result. The correct interpretation is difficult due to the small size of the DNA fragments. In this study we designed a new set of primers for amplification of a 144bp fragment that facilitates the reading of the result. In addition, we optimized the PCR reaction to allow amplification from a single hair bulb, added directly into the PCR mix without previous treatment. This improved method was used to genotype 165 sows and boars used in a breeding program. Forty-nine percent of the animals had the NN genotype, whereas 50% were Nn and only 1% was nn.