Overlap Extension PCR Research Articles

Rigidifying Flexible Sites: A Promising Strategy to Improve Thermostability of Lysophospholipase From Pyrococcus abyssi.

High thermostability of the enzymes is one of the distinguishing characteristics that increase their industrial utility. In the current research work, rigidifying the flexible amino acid residues of a lysophospholipase (Pa-LPL) from Pyrococcus abyssi was used as a protein engineering approach to improve its thermostability. A truncated variant of Pa-LPL (t-LPL∆12) was constructed by trimming its 12 amino acid residues (50-61) through overlap extension PCR. The truncated enzyme worked optimally at 65°C and pH 6.5 with remarkable thermostability at 65°C-85°C. In comparison to wild-type Pa-LPL, 5.8 and 1.2-fold increase in half-life (t1/2) of t-LPL∆12 was observed at 65 (optimum temperature) and 95°C, respectively. The activity of t-LPL∆12 was stimulated by 1 mM Cu2+ followed by Ca2+, Ni2+, Co2+, and Mg2+. Both substrate docking and experimental results indicated that the truncated enzyme could hydrolyze a variety of p-nitrophenyl esters. Km, Vmax, and Kcat for enzymatic hydrolysis of p-nitrophenyl butyrate were calculated to be 1 ± 0.087 mM, 1456 ± 36.474 U/mg, and 1.397 × 1011 min-1, respectively. In short, broad substrate specificity and thermostability of t-LPL∆12 are some of the distinctive features that make it an ideal candidate for degumming of vegetable oils.

Glycine-replaced epinecidin-1 variant bestows better stability and stronger antimicrobial activity against a range of nosocomial pathogenic bacteria.

Epinecidin-1 (epi-1), an antimicrobial peptide first identified in marine grouper fish, has multifunctional bioactivities. The present study aims to improve its therapeutic potential via structural modifications that could enhance its antimicrobial activity and stability. To achieve it, we replaced glycine and the first histidine in the parent epi-1 with lysine, which resulted in a peptide with a repeating KXXK motif and improved physiochemical properties related to antimicrobial activity. This modified peptide, referred to as glycine-to-lysine replaced-epi-1, also gained stability and a twofold increase in helical propensity. To produce the active peptide, overlap extension PCR was employed to generate the gene of GK-epi-1 via site-directed mutagenesis, which was then cloned into the pET-32a vector and expressed as a recombinant fusion protein in Escherichia coli C43 (DE3) strain. The recombinant protein was purified and digested with enterokinase to release the active peptide fragment, which was then evaluated for antimicrobial activity and stability. The lysine substitution led to an enhancement in broad-spectrum antimicrobial activity against a wide range of nosocomial pathogenic bacteria.

High-fidelity CRISPR/Cas12a dual-crRNA screening reveals novel synergistic interactions in hepatocellular carcinoma.

: CRISPR/Cas12a-based combinational screening has shown remarkable potential for identifying genetic interactions. Here, we describe an innovative method for combinational genetic screening with rapid construction of a dual-CRISPR RNA (crRNA) library using gene splicing through overlap extension PCR (SOE PCR) and the adoption of CeCas12a, which we previously identified with strict PAM recognition and low off-targeting to guarantee fidelity and efficiency. The custom-pooled SOE crRNA array (SOCA) library for double-knockout screening could be conveniently constructed in the laboratory for widespread use, and the CeCas12a-mediated high-fidelity screen displayed good performance even under a negative selection screen. By designing a SOCA dual-crRNA library that covered most of the kinase and metabolism-associated gene targets of FDA-approved drugs implicated in hepatocellular carcinoma (HCC) tumourigenesis, novel cross-talk between the two gene sets was negatively selected to inhibit HCC cell growth in vitro and in vivo and was validated using virtual double-knockdown screening based on TCGA databases. Thus, this rapid, efficient and high-fidelity double-knockout screening system is promising for systemically identifying potential genetic interactions between multiple gene sets or combinations of FDA- approved drugs for clinical translational medicine in the future.

Cloning, Expression, and Bioinformatics Modeling of Human Papillomavirus Type 52 L1/L2 Chimeric Protein in Escherichia coli BL21 (DE3)

Human papillomavirus (HPV) L1 major capsid protein generates a highly immunogenic virus like particles (VLPs), which have been used as the main component of its prophylactic vaccine. However, the neutralizing antibodies against L1 VLPs are mostly type specific and may not be effective to prevent infection from different strains of HPV. On the other hand, HPV L2 minor capsid protein has low antigenic variation, thus can induce cross-neutralization. This study aims to obtain HPV 52 L1/L2 chimeric protein, which is designed based on HPV type 52 as one of the most circulated high-risk types in Indonesia, to develop a broad-spectrum HPV vaccine. Substitution of HPV 52 H4 helix L1 region with an HPV 52 L2 epitope was carried out using overlap extension PCR. HPV 52 L1/L2 chimeric gene was constructed into pET-SUMO expression vector and expressed in Escherichia coli BL21 (DE3). Bioinformatics modeling suggested that L2 epitope was located inside of the loop region in monomer form, and on the contrary, it was located outside of the pentamer surface. Furthermore, B cell and T cell epitopes predictions were conducted using Immune Epitope Database (IEDB) analysis. The B cell epitopes prediction revealed eleven potential epitopes, whereas the T cell epitopes prediction showed seven potential epitopes for each MHC class I and MHC class II. This study showed that HPV 52 L1/L2 chimeric protein has the potential to induce cross-neutralizing antibodies and can be developed as a promising candidate for a new HPV vaccine.

Unraveling the peculiarities and development of novel inhibitors of leishmanial arginyl-tRNA synthetase.

Aminoacylation by tRNA synthetase is a crucial part of protein synthesis and is widely recognized as a therapeutic target for drug development. Unlike the arginyl-tRNA synthetases (ArgRSs) reported previously, here, we report an ArgRS of Leishmania donovani (LdArgRS) that can follow the canonical two-step aminoacylation process. Since a previously uncharacterized insertion region is present within its catalytic domain, we implemented the splicing by overlap extension PCR (SOE-PCR) method to create a deletion mutant (ΔIns-LdArgRS) devoid of this region to investigate its function. Notably, the purified LdArgRS and ΔIns-LdArgRS exhibited different oligomeric states along with variations in their enzymatic activity. The full-length protein showed better catalytic efficiency than ΔIns-LdArgRS, and the insertion region was identified as the tRNA binding domain. In addition, a benzothiazolo-coumarin derivative (Comp-7j) possessing high pharmacokinetic properties was recognized as a competitive and more specific inhibitor of LdArgRS than its human counterpart. Removal of the insertion region altered the mode of inhibition for ΔIns-LdArgRS and caused a reduction in the inhibitor's binding affinity. Both purified proteins depicted variances in the secondary structural content upon ligand binding and thus, thermostability. Apart from the trypanosomatid-specific insertion and Rossmann fold motif, LdArgRS revealed typical structural characteristics of ArgRSs, and Comp-7j was found to bind within the ATP binding pocket. Furthermore, the placement of tRNAArg near the insertion region enhanced the stability and compactness of LdArgRS compared to other ligands. This study thus reports a unique ArgRS with respect to catalytic as well as structural properties, which can be considered a plausible drug target for the derivation of novel anti-leishmanial agents.

Preparation and immunogenicity evaluation of C-HapS-P6 fusion protein vaccine against nontypeable Haemophilus influenzae in mice

Nontypeable Haemophilus influenzae (NTHi) is the dominant pathogen in several infectious diseases. Currently the use of antibiotics is the main intervention to prevent NTHi infections, however with the emergence of drug resistant strains, it has compromised the treatment of respiratory infections with antibiotics. Therefore there is an urgent need to develop a safe and effective vaccine to prevent NTHi infections. We investigate the potential of C-HapS-P6 fusion protein as a vaccine for treating NTHi in murine models. PGEX-6P2/C-HapS-P6 fusion gene was constructed using overlap extension polymerase chain reaction. The recombined plasmid was transformed into Escherichia coli for protein expression. The mice were subjected to intraperitoneal immunization using purified antigens. Immunoglobulin (Ig) G in serum samples and IgA in nasal and lung lavage fluids were analyzed using enzyme-linked immunosorbent assay. Cytokine release and proliferation capacity of splenic lymphocytes in response to antigens were measured in vitro. The protective effect of the C-HapS-P6 protein against NTHi infection was evaluated by NTHi count and histological examination. The data showed that the C-HapS-P6 fusion protein increased significantly the levels of serum IgG and nasal and lung IgA, and promoted the release of interleukin (IL)-2, interferon-ϒ, IL-4, IL-5, and IL-17 and the proliferation of splenic lymphocytes compared with C-HapS or P6 protein treatment alone. Moreover, C-HapS-P6 effectively reduced the NTHi colonization in the nasopharynx and lungs of mice. In conclusion, our results demonstrated that the C-HapS-P6 fusion protein vaccine can significantly enhance humoral and cell immune responses and effectively prevent against NTHi infection in the respiratory tract in murine models.

Techniques for Genetic Manipulation.

In the last few decades, molecular techniques and genetic modification have been used in genotype and phenotype studies of S. suis. Genomic modification of S. suis requires DNA acquisition and its stable insertion into the chromosome by allelic exchange. In this chapter, we described two techniques for the preparation of genomic constructs (cloning and overlapping extension PCR) and for DNA uptake (electroporation and transformation). The protocols are accompanied with examples. All described protocols were successful on our hands with the reference S. suis strain P1/7.

Design and Construction of a Designed Ankyrin Repeat Protein (DARPin) Display Library.

Protein display systems are powerful techniques used to identify protein molecules that bind with high affinity to target proteins of interest. The initial challenge in implementing a display system is the construction of a high-diversity naïve library. Here, we describe the methods to generate a designed ankyrin repeat protein (DARPin) display library using degenerate oligonucleotides. Specifically described is the construction of a single DARPin repeat module by overlap extension PCR, concatenation of the module by restriction enzyme digestion and ligation, and incorporation of the concatenated modules into a full-length DARPin sequence in a bacterial cloning or display vector containing the hydrophilic N- and C-terminal capping domains. Protocols for PCR amplification of DARPin sequences to estimate diversity of naïve and enriched libraries via next-generation sequencing are included, as is a simple Linux-based program for analysis of naïve and enriched sequences. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of a single DARPin repeat by overlap extension PCR Basic Protocol 2: Concatenation of DARPin repeats Basic Protocol 3: Ligation of internal repeats into cloning/display vector containing N- and C-terminal capping repeats Basic Protocol 4: Estimation of library size and diversity by next-generation sequencing (NGS) Basic Protocol 5: NGS analysis of naïve and enriched libraries.

Characterization of Type VI secretion system in Edwardsiella ictaluri.

Edwardsiella ictaluri is a Gram-negative facultative intracellular fish pathogen causing enteric septicemia of catfish (ESC). While various secretion systems contribute to E. ictaluri virulence, the Type VI secretion system (T6SS) remains poorly understood. In this study, we constructed 13 E. ictaluri T6SS mutants using splicing by overlap extension PCR and characterized them, assessing their uptake and survival in channel catfish (Ictalurus punctatus) peritoneal macrophages, attachment and invasion in channel catfish ovary (CCO) cells, in vitro stress resistance, and virulence and efficacy in channel catfish. Among the mutants, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO exhibited reduced replication inside peritoneal macrophages. EiΔevpM, EiΔevpN, and EiΔevpO showed significantly decreased attachment to CCO cells, while EiΔevpN and EiΔevpO also displayed reduced invasion of CCO cells (p < 0.05). Overall, T6SS mutants demonstrated enhanced resistance to oxidative and nitrosative stress in the nutrient-rich medium compared to the minimal medium. However, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO were susceptible to oxidative stress in both nutrient-rich and minimal medium. In fish challenges, EiΔevpD, EiΔevpE, EiΔevpG, EiΔevpJ, and EiΔevpK exhibited attenuation and provided effective protection against E. ictaluri wild-type (EiWT) infection in catfish fingerlings. However, their attenuation and protective efficacy were lower in catfish fry. These findings shed light on the role of the T6SS in E. ictaluri pathogenesis, highlighting its significance in intracellular survival, host cell attachment and invasion, stress resistance, and virulence. The attenuated T6SS mutants hold promise as potential candidates for protective immunization strategies in catfish fingerlings.

Isolation and characterization of a novel single-chain variable fragment (scFv) against Lymphocyte function-associated antigen-1 (LFA-1) using phage display method.

Lymphocyte function-associated antigene-1 (LFA-1) is a well-described integrin found on lymphocytes and other leukocytes, which is known to be overexpressed in leukemias and lymphomas. This receptor plays a significant role in immune responses such as T-cell activation, leukocyte cell-cell interactions, and trafficking of leukocyte populations. Subsequently, binders of LFA-1 emerge as potential candidates for cancer and autoimmune therapy. This study used the phage display technique to construct and characterize a high-affinity single-chain fragment variable (scFv) antibody against LFA-1. After expression, purification, dialysis, and concentration of the recombinant LFA-1 protein, four female BALB/c mice were immunized, splenocyte's mRNA was extracted, and cDNA was synthesized. A scFv library was constructed by linking the amplified VH/Vκ fragments through a 72-bp linker using SOEing PCR. Next, the scFv gene fragments were cloned into the pComb-3XSS phagemid vector; thus, the phage library was developed. The selection process involved three rounds of phage-bio-panning, polyclonal, and monoclonal phage ELISA. AF17 was chosen and characterized among the positive clones through SDS-PAGE, Western blotting, indirect ELISA, and in-silico analyses. The results of the study showed the successful construction of a high-affinity scFv library against LFA-1. The accuracy of the AF17 production and its ability to bind to the LFA-1 were confirmed through SDS-PAGE, Western blot, and ELISA. This study highlights the potential application of the high-affinity AF17 against LFA-1 for targeting T lymphocytes for therapeutic purposes.

Functional characterization of Clonorchis sinensis choline transporter.

Clonorchis sinensis is commonly found in East Asian countries. Clonorchiasis is prevalent in these countries and can lead to various clinical symptoms. In this study, we used overlap extension polymerase chain reaction (PCR) and the Xenopus laevis oocyte expression system to isolate a cDNA encoding the choline transporter of C. sinensis (CsChT). We subsequently characterized recombinant CsChT. Expression of CsChT in X. laevis oocytes enabled efficient transport of radiolabeled choline, with no detectable uptake of arginine, α-ketoglutarate, p-aminohippurate, taurocholate, and estrone sulfate. Influx and efflux experiments showed that CsChT-mediated choline uptake was time- and sodium-dependent, with no exchange properties. Concentration-dependent analyses of revealed saturable kinetics consistent with the Michaelis-Menten equation, while nonlinear regression analyses revealed a Km value of 8.3 μM and a Vmax of 61.0 pmol/oocyte/h. These findings contribute to widen our understanding of CsChT transport properties and the cascade of choline metabolisms within C. sinensis.

Gibson assembly interposition improves amplification efficiency of long DNA and multifragment overlap extension PCR.

For difficult overlap extension PCR, a Gibson assembly process was inserted between the two PCR rounds to facilitate the formation of complete gene templates at a moderate temperature. That is, after amplifying each DNA fragment, they were preluded by a Gibson assembly process in equal proportion. Then, the assembled mixture was used as a template for the second PCR round. This idea was tested and verified by taking the cloning example of a single and a double site mutation of the retinoblastoma gene. This scheme associates overlap extension PCR with Gibson assembly exquisitely, significantly improving gene amplification efficiency, particularly in the fusion of long genes and multifragments using overlap extension PCR.

Genetically engineered deletion in the N-terminal region of nifA1 in R. capsulatus to enhance hydrogen production

NifA is the primary activator of nitrogenase, and the N-terminal domain of nifA is sensitive to ammonium concentration. In this work, a mutant Rhodobacter capsulatus ZX01 with a genetically engineered deletion in the N-terminal region of nifA1 was constructed by employing overlap extension PCR to mitigate the inhibition of ammonium on nitrogenase expression in photosynthetic bacteria. The effects of different ammonium ion concentrations on the growth and photo-fermentative hydrogen production performance of wild-type strain R. capsulatus SB1003 and mutant ZX01 with glucose and volatile fatty acids as the carbon sources were studied, respectively. When the ratio of NH4+-N was 20% and 30%, the hydrogen yield of the mutant ZX01 was enhanced by 14.8% and 20.9% compared with that of R. capsulatus SB1003 using 25 mM acetic acid and 34 mM butyric acid as the carbon source, respectively. In comparison, using 30 mM glucose as the carbon source, the hydrogen yield of ZX01 was increased by 17.7% and 22.2% compared with that of R. capsulatus SB1003 when the ratio of NH4+-N was 20% and 30%, and the nitrogenase activity of ZX01 was also enhanced by 38.0% and 47.6%, respectively. When using 10 mM NH4+ as a single nitrogen source, ZX01 showed a 2.6-fold increase in H2 production. These results indicated that ZX01 demonstrated higher ammonium tolerance and better hydrogen production performance than the wild-type. The deletion in the N-terminal region of nifA1 could partially de-repress the nitrogenase activity inhibited by ammonium.

Biological Activity of Optimized Codon Bovine Type III Interferon Expressed in Pichia pastoris.

Type III interferons (IFN-λs) exhibit potent antiviral activity and immunomodulatory effects in specific cells. Nucleotide fragments of the bovine ifn-λ (boifn-λ) gene were synthetized after codon optimization. The boifn-λ gene was then amplified by splicing using overlap extension PCR (SOE PCR), resulting in the serendipitous acquisition of the mutated boIFN-λ3V18M. The recombinant plasmid pPICZαA-boIFN-λ3/λ3V18M was constructed, and the corresponding proteins were expressed in Pichia pastoris with a high-level extracellular soluble form. Dominant expression strains of boIFN-λ3/λ3V18M were selected by Western blot and ELISA and cultured on a large scale, and the recombinant proteins purified by ammonium sulfate precipitation and ion exchange chromatography yielded 1.5g/L and 0.3 g/L, with 85% and 92% purity, respectively. The antiviral activity of boIFN-λ3/λ3V18M exceeded 106 U/mg, and they were neutralized with IFN-λ3 polyclonal antibodies, were susceptible to trypsin, and retained stability within defined pH and temperature ranges. Furthermore, boIFN-λ3/λ3V18M exerted antiproliferative effects on MDBK cells without cytotoxicity at 104 U/mL. Overall, boIFN-λ3 and boIFN-λ3V18M did not differ substantially in biological activity, except for reduced glycosylation of the latter. The development of boIFN-λ3 and comparative evaluation with the mutant provide theoretical insights into the antiviral mechanisms of boIFN-λs and provide material for therapeutic development.

SOEing PCR/Docking Optimization of Protein A-G/scFv-Fc-Bioconjugated Au Nanoparticles for Interaction with Meningitidis Bacterial Antigen

Recent advances in the use of gold nanoparticles (Au NPs)/antibody conjugations in nanomedicine have increased the need to optimize the synthesis conditions and surface functionalization of Au NPs. In this study, a home-made Neisseria meningitidis recombinant antibody (scFv-Fc) was developed by connecting the fragment crystallizable (Fc) region of a human antibody with a mouse recombinant antibody (single-chain variable fragment antibody (scFv)) and characterized using the SOEing PCR technique. Then, an optimized gold coating agent for the scFv-Fc/Au NP conjugation (i.e., the citrate agent) was found among three common agents (citrate, allylamine hydrochloride, and polyvinyl alcohol) with different surface charges (negative, positive, and neutral, respectively). Moreover, the stability of the scFv-Fc/protein A-G in the presence of a N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) linker was investigated using the docking method. It was found that the designed scFv-Fc/protein A-G/SPDP/citrate recombinant antibody showed optimized bottom-on conjugation of the protein A-G with the improved scFv-Fc/Au NPs, enabling a suitable interaction with the Neisseria meningitidis bacterial antigen.

Deletion of FTL_1199 to determine the role of this gene in erythrocyte invasion by Francisella tularensis.

Francisella tularensis is a bacterium that induces the zoonotic disease tularemia. In the course of infection, F. tularensis bacteria invade erythrocytes, a phenomenon that heightens the colonization of ticks after a blood meal. To better understand the mechanism of erythrocyte invasion, we hypothesized that transcription of bacterial genes significant in erythrocyte invasion would be upregulated upon exposure to these host cells. An RNA-seq unveiled that transcription of 7% of F. tularensis genes augment when in erythrocyte presence. Of these, we pinpointed a putative transcriptional regulator, FTL_1199. The goal was to delete FTL_1199 in F. tularensis LVS. SOE PCR amplified and duplicated the up and downstream regions of the target gene in tandem into a shuttle vector that is insecure within F. tularensis. This newly generated plasmid, pDEL1199, was mobilized inside of F. tularensis by conjugation. Merodiploid strains generated by homologous recombination were isolated and transformed with pGUTS. Expression of I-Sce1 within the merodiploid produces a double-stranded break. This breakage resulted in a second recombination that either ensued to wild-type or deletion of FTL_1199 deduced through a PCR. Finally, in DFTL_1199 strains, pGUTS was cured by successive cultivation in the absence of selection followed by replica-plating on chocolate II agar ± kanamycin. Gentamicin protection assays showed reduced levels of erythrocyte invasion for F. tularensis DFTL_1199 compared to wild type bacteria. However, complementation of FTL_1199 to the deletion mutant restored this strain’s ability to invade red blood cells. These findings demonstrate that FTL_1199 is important for erythrocyte invasion by F. tularensis.&#x0D; &#x0D; (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence, R15HL14735 from NHLBI, and funds from the NASA West Virginia Space Grant Consortium).

Identification and genetic characterisatin of cathepsin L in Demodex.

Owing to difficulties in obtaining functional gene sequences, molecular pathogenic mechanisms in Demodex have been understudied. In this study, overlap extension PCR was used to obtain the sequences of cathepsin L (CatL), a pathogenicity-related gene, to provide a foundation for subsequent functional research. Demodex folliculorum and Demodex brevis mites were obtained from the face skin of Chinese individuals, and Demodex canis mites were isolated from the skin lesions of a dog. RNA was extracted and used to synthesise double-stranded cDNA. PCR amplification, cloning, sequencing, and bioinformatics analysis of CatL were performed. CatL gene sequences of 1005, 1008, and 1008bp were successfully amplified for D. brevis, D. folliculorum, and D. canis, respectively. These sequences showed 99.9 or 100% identity with templates previously obtained by RNA-seq. The Maximum Likelihood (ML) phylogenetic tree showed that D. folliculorum clustered with D. canis first, then with D. brevis, and finally with other Acariformes mite species. The three Demodex species had nine similar motifs to those of Sarcoptes scabies, Dermatophagoides pteronyssinus, and Dermatophagoides farinae, and motifs 10-13 were valuable for identification. CatL proteins of Demodex species were predicted to be approximately 38kDa, be located in lysosomes, have a signal peptide but no transmembrane region, and have two functional domains, I29 and Pept_C1. However, interspecific differences were observed in secondary and tertiary protein structures. In conclusion, we successfully obtained CatL sequences of three Demodex species by overlap extension PCR, which creates conditions for further pathogenic mechanism studies.

CRISPR/Cas9-assisted ssDNA recombineering for site-directed mutagenesis and saturation mutagenesis of plasmid-encoded genes

Site-directed and saturation mutagenesis are critical DNA methodologies for studying protein structure and function. For plasmid-based gene mutation, PCR and overlap-extension PCR involve tedious cloning steps. When the plasmid size is large, PCR yield may be too low for cloning; and for saturation mutagenesis of a single codon, one experiment may not enough to generate all twenty coding variants. Oligo-mediated recombineering sidesteps the complicated cloning process by homologous recombination between a mutagenic oligo and its target site. However, the low recombineering efficiency and inability to select for the recombinant makes it necessary to screen a large number of clones. Herein, we describe two plasmid-based mutagenic strategies: CRISPR/Cas9-assisted ssDNA recombineering for site-directed mutagenesis (CRM) and saturation mutagenesis (CRSM). CRM and CRSM involve co-electroporation of target plasmid, sgRNA expression plasmid and mutagenic oligonucleotide into Escherichia coli cells with induced expression of λ-Red recombinase and Cas9, followed by plasmid extraction and characterization. We established CRM and CRSM via ampicillin resistance gene repair and mutagenesis of N-acetyl‑D‑neuraminic acid aldolase. The mutational efficiency was between 80 and 100% and all twenty amino acid coding variants were obtained at a target site via a single CRSM strategy. CRM and CRSM have the potential to be general plasmid-based gene mutagenesis tools.

Research experimental design for the construction and identification of the pGEX-BCKD-E4A recombinant point-mutant plasmid.

Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that eventually develops into cirrhosis and even liver cancer. In recent years, the incidence rate has been increasing, and the early diagnosis and treatment of PBC are crucial. In the early diagnosis method of PBC, anti-mitochondrial antibodies (AMAs) are an important diagnostic basis, especially the M2 subtype (AMA-M2) with almost 100% specificity. We selected the BCOADC-E2 protein, a mitochondrial autoantigen that reacts specifically with AMA-M2 antibodies, and carried out DNA recombination and protein mutation experiments by cloning in vitro the homologous target gene sequence BCKD that expresses the antigenic epitope of BCOADC-E2 protein, to provide experience for later exploring the effect of mutations of amino acids around the lysine in the active center of BCOADC-E2 protein on its specific binding to AMA-M2, and to lay the foundation for determining the key amino acids of BCOADC-E2 for the diagnosis and treatment of PBC. In addition, we apply this scientific research content to graduate course teaching. Experimental technology of microbial molecular ecology is a course with the cross-integration of multidisciplinary knowledge and experimental skills offered at our college since 2018. This article derives from the part of this course on the construction of recombinant plasmids. The students first constructed the recombinant plasmid pGEX-BCKD using the vector plasmid pGEX-4T1 and the target gene fragment BCKD provided by the laboratory and used this as a template to construct the pGEX-BCKD-E4A point mutation plasmid by the overlap extension PCR (SOE PCR) technique to achieve the effect of mutating the fifth amino acid glutamate in front of lysine, the active centre of the BCOADC-E2 lipid acyl binding domain, to alanine for subsequent studies. Through the research experiment, combining theoretical knowledge and experimental operation, we aim to deepen the student's understanding of DNA recombination technology, let them feel the practical application prospect of experimental technology, stimulate students' interest in professional knowledge learning, and cultivate students' scientific thinking and innovation consciousness. We examined the quality of the teaching through the process and summative evaluation of the students. In this study, the students successfully completed the construction of pGEX-BCKD-E4A point mutant plasmid, and the average test score increased from 40.4% before teaching to 91.1%. The teaching effect was remarkable. This kind of research experimental teaching mode has good application prospects, and other education and teachers can refer to and reference it.

Construction and Rescue of a Minigenome System for Lentogenic D58 Strain of Avian orthoavulavirus 1 (Newcastle Disease Virus)

Background: In reverse genetics studies, minigenomes of negative sense RNA viruses are used to model the process of virus replication and evaluate the virus rescue system in the course of construction of recombinant virus. In the current study, a minigenome (MG) rescue system for lentogenic D58 vaccine strain of Avian orthoavulavirus 1 (AOAV-1) (Newcastle disease virus) was developed to establish a reverse genetic system for this strain. Methods: The minigenome was constructed by assembling the trailer and leader regions of AOAV-1 D58 strain flanking the reporter gene, Aequorea coerulescens green fluorescence protein (AcGFP) in a modified transcription vector. Construction of this Minigenome was done using a simple restriction free technique-Splicing by Overlap Extension PCR. The constructed minigenome was evaluated for the reporter gene expression by transfection in T7 RNA polymerase expressing BSR/T7 cell line. Further, the replication and packaging ability of the constructed minigenome were also analyzed. Result: The reporter gene expression was confirmed in BSR/T7 cells which simulates the virus replication process and verifies the virus rescue system. In addition, this proves the applicability of restriction free cloning by Splicing by Overlap Extension (SOE) PCR for precise construction of the minigenome. The developed minigenome system can be used as a potential tool in the reverse genetic rescue of AOAV-1 D58 strain for the development of marker vaccine.