PD-L1 targeted peptide demonstrates potent antitumor and immunomodulatory activity in cancer immunotherapy.

Background: In recent years, immunotherapy has been emerging as a promising alternative therapeutic method for cancer patients, offering potential benefits. The expression of PD-L1 by tumors can inhibit the T-cell response to the tumor and allow the tumor to evade immune surveillance. To address this issue, cancer immunotherapy has shown promise in disrupting the interaction between PD-L1 and its ligand PD-1. Methods: We used mirror-image phage display technology in our experiment to screen and determine PD-L1 specific affinity peptides (PPL-C). Using CT26 cells, we established a transplanted mouse tumor model to evaluate the inhibitory effects of PPL-C on tumor growth in vivo. We also demonstrated that PPL-C inhibited the differentiation of T regulatory cells (Tregs) and regulated the production of cytokines. Results: In vitro, PPL-C has a strong affinity for PD-L1, with a binding rate of 0.75 µM. An activation assay using T cells and mixed lymphocytes demonstrated that PPL-C inhibits the interaction between PD-1 and PD-L1. PPL-C or an anti-PD-L1 antibody significantly reduced the rate of tumor mass development in mice compared to those given a control peptide (78% versus 77%, respectively). The results of this study demonstrate that PPL-C prevents or retards tumor growth. Further, immunotherapy with PPL-C enhances lymphocyte cytotoxicity and promotes proliferation in CT26-bearing mice. Conclusion: PPL-C exhibited antitumor and immunoregulatory properties in the colon cancer. Therefore, PPL-C peptides of low molecular weight could serve as effective cancer immunotherapy.

Immunoinformatic Identification of Multiple Epitopes of gp120 Protein of HIV-1 to Enhance the Immune Response against HIV-1 Infection.

Acquired Immunodeficiency Syndrome is caused by the Human Immunodeficiency Virus (HIV), and a significant number of fatalities occur annually. There is a dire need to develop an effective vaccine against HIV-1. Understanding the structural proteins of viruses helps in designing a vaccine based on immunogenic peptides. In the current experiment, we identified gp120 epitopes using bioinformatic epitope prediction tools, molecular docking, and MD simulations. The Gb-1 peptide was considered an adjuvant. Consecutive sequences of GTG, GSG, GGTGG, and GGGGS linkers were used to bind the B cell, Cytotoxic T Lymphocytes (CTL), and Helper T Lymphocytes (HTL) epitopes. The final vaccine construct consisted of 315 amino acids and is expected to be a recombinant protein of approximately 35.49 kDa. Based on docking experiments, molecular dynamics simulations, and tertiary structure validation, the analysis of the modeled protein indicates that it possesses a stable structure and can interact with Toll-like receptors. The analysis demonstrates that the proposed vaccine can provoke an immunological response by activating T and B cells, as well as stimulating the release of IgA and IgG antibodies. This vaccine shows potential for HIV-1 prophylaxis. The in-silico design suggests that multiple-epitope constructs can be used as potentially effective immunogens for HIV-1 vaccine development.

Exosomes multifunctional roles in HIV-1: insight into the immune regulation, vaccine development and current progress in delivery system.

Human Immunodeficiency Virus (HIV-1) is known to establish a persistent latent infection. The use of combination antiretroviral therapy (cART) can effectively reduce the viral load, but the treatment can be costly and may lead to the development of drug resistance and life-shortening side effects. It is important to develop an ideal and safer in vivo target therapy that will effectively block viral replication and expression in the body. Exosomes have recently emerged as a promising drug delivery vehicle due to their low immunogenicity, nanoscale size (30-150nm), high biocompatibility, and stability in the targeted area. Exosomes, which are genetically produced by different types of cells such as dendritic cells, neurons, T and B cells, epithelial cells, tumor cells, and mast cells, are designed for efficient delivery to targeted cells. In this article, we review and highlight recent developments in the strategy and application of exosome-based HIV-1 vaccines. We also discuss the use of exosome-based antigen delivery systems in vaccine development. HIV-1 antigen can be loaded into exosomes, and this modified cargo can be delivered to target cells or tissues through different loading approaches. This review also discusses the immunological prospects of exosomes and their role as biomarkers in disease progression. However, there are significant administrative and technological obstacles that need to be overcome to fully harness the potential of exosome drug delivery systems.

Identification of candidate hub genes correlated with the pathogenesis, diagnosis, and prognosis of prostate cancer by integrated bioinformatics analysis.

Background: Prostate cancer (PCa) has the second highest morbidity and mortality rates in men. Concurrently, novel diagnostic and prognostic biomarkers of PCa remain crucial. Methods: This study utilized integrated bioinformatics method to identify and validate the potential hub genes with high diagnostic and prognostic value for PCa. Results: Four Gene Expression Omnibus (GEO) datasets including 123 PCa samples and 76 normal samples were screened and a total of 368 differentially expressed genes (DEGs), including 120 up-regulated DEGs and 248 down-regulated DEGs, were identified. Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEGs were majorly enriched in focal adhesion, chemical carcinogenesis, drug metabolism, and cytochrome P450 pathways. Then, 11 hub genes were identified from the protein-protein interaction (PPI) network of the DEGs; 7 of the 11 genes showed the ability of distinguishing PCa from normal prostate based on receiver operating characteristic (ROC) curve analysis. And 5 of the 11 genes were correlated with clinical attributes. Lower CAV1, KRT5, SNAI2 and MYLK expression level were associated with higer Gleason score, advanced pathological T stage and N stage. Lower KRT5 and MYLK expression level were significantly correlated with poor disease-free survival, and lower KRT5 and PTGS2 expression level were significantly related to biochemical recurrence (BCR) status of PCa patients. Conclusions: In conclusion, CAV1, KRT5, SNAI2, and MYLK show potential clinical diagnostic and prognostic value and could be used as novel candidate biomarkers and therapeutic targets for PCa.

Six Novel Biomarkers for Diagnosis and Prognosis of Esophageal squamous cell carcinoma: validated by scRNA-seq and qPCR.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide. ESCC has a generally poor prognosis and there is a lack of available biomarkers for diagnosis and prognosis. The aim of the study was to identify novel biomarkers for ESCC. We screened the overlapping differentially expressed genes (DEGs) acquired from six Gene Expression Omnibus (GEO) ESCC datasets and The Cancer Genome Atlas (TCGA) ESCC datasets. Subsequently, protein-protein interaction network analysis was performed to identify the key hub genes. Then, Kaplan Meier survival and receiver operating curve (ROC) analysis were utilized to clarify the diagnostic and prognostic role of these hub genes. The UALCAN database, single cell RNA sequencing (scRNA-seq) and real-time quantitative PCR (qPCR) were performed to confirm the expression levels of identified hub genes. Finally, immune infiltration analysis was conducted to investigate the role of these genes in the pathogenesis of ESCC. The results showed that PBK, KIF2C, NUF2, KIF20A, RAD51AP1, and DEPDC1 effectively distinguish ESCC tissues from normal samples, and all of them were significantly correlated with overall survival. The results of scRNA-seq and qPCR indicated that the expression levels of hub genes in ESCC were significantly higher than in normal cells or tissues. Further immune infiltration analysis showed that infiltration of dendritic cells was significantly negatively correlated with PBK, KIF2C, NUF2, RAD51AP1, and DEPDC1 expression levels. In conclusion, our results suggest that PBK, KIF2C, NUF2, KIF20A, RAD51AP1 and DEPDC1 are all potential biomarkers for ESCC diagnosis and prognosis may also be potential therapeutic targets for ESCC.

The Diagnostic Value of Antibodies Against Citrullinated or Carbamylated Fibrinogen in Rheumatoid Arthritis.

BACKGROUND: This study aimed to evaluate the overall diagnostic value of citrullinated or carbamylated fibrinogen antibodies in patients with rheumatoid arthritis (RA). METHODS: Serum samples collected from 114 patients with established RA, 143 patients with non-RA diseases, and 200 healthy controls were tested by ELISA for citrullinated fibrinogen (Cit-fib), carbamylated fibrinogen (Ca-fib), and chimeric fibrinogen a/b chain citrullinated peptides (CFABCP). Diagnostic indexes and correlations with titers were calculated, cross reactivities of Cit-fib, Ca-fib, and CFABCP were assessed by competition experiments. RESULTS: With a cutoff ensuring 98% specificity for RA patients versus healthy controls, the sensitivities of Cit-fib and Ca-fib are 66.67% and 24.6%, respectively, while the sensitivity of CFABCP was 74.56%. Cit-fib, Ca-fib, and CFABCP can inhibit reciprocally in competition experiments. As for non-RA patients, the positive rate of Ca-fib was higher than that of Cit-fib and CFABCP. CONCLUSIONS: Citrullination and carbamylation of fibrinogen both have a role in RA diagnosing, but citrullination is better. The recombination of peptides, CFABCP, has high specificity and considerable sensitivity for diagnosis for RA patients.

Cellular UAP56 interacts with the HBx protein of the hepatitis B virus and is involved in viral RNA nuclear export in hepatocytes.

UAP56 is an essential factor in eukaryotic pre-mRNA splicing and mRNA export. Many viruses require cellular RNA export factors to efficiently export viral RNA. However, the mechanisms behind hepatitis B virus (HBV) RNA splicing and nuclear export remain poorly understood. Here, our data show that UAP56 interacts with the HBx protein. Moreover, we demonstrate that the Q-motif of UAP56, which regulates RNA-binding and helicase activity, is essential for the interaction of UAP56 with HBx. Both knockdown of UAP56 and deficiency of HBx impaired cytoplasmic accumulation of HBV RNA transcripts, whereas knockdown of UAP56 also reduced the level of HBV pregenomic RNA splicing variants. In addition, knockdown of Nxf1 induced HBV RNA nuclear accumulation. These findings provide unique insights into the mechanistic details of HBV RNA export and splicing.

Meta-analysis: diagnostic accuracy of the citrullinated peptides derived from fibrinogen and vimentin in rheumatoid arthritis.

OBJECTIVES: Anticitrullinated protein/peptide antibodies (ACPAs) have shown valuable effects in the diagnosis of rheumatoid arthritis (RA). Both fibrinogen and vimentin are significant antigens of ACPAs. This study evaluated the diagnostic performance of fibrinogen and vimentin peptides in RA. METHODS: We searched the PubMed, Embase, and Cochrane Library databases for studies published in English until January 2019 that evaluated the utility of the peptides of both vimentin and fibrinogen. The bivariate mixed-effects model and summary receiver operating characteristic (SROC) curve were used to estimate sensitivity and specificity across studies. RESULTS: Seven peptides from 19 studies were included. The pooled sensitivities of Fibα36-50, Fibα563-583, Fibα580-600, Fibα621-635, Fibß36-52, Fibß60-74, and Vim60-75 were 35%, 41%, 18%, 26%, 0.53%, 57%, and 44%, respectively. The pooled specificities of Fibα36-50, Fibα563-583, Fibα580-600, Fibα621-635, Fibß36-52, Fibß60-74, and Vim60-75 were 97%, 98%, 98%, 98%, 97%, 98%, and 98%, respectively. The SROC areas under the curve (AUCs) of Fibα36-50, Fibα563-583, Fibα580-600, Fibα621-635, Fibß36-52, Fibß60-74, and Vim60-75 were 0.92, 0.85, 0.64, 0.82, 0.91, 0.96, and 0.86, respectively. CONCLUSION: Both fibrinogen and vimentin peptides have a high specificity but a relatively low sensitivity in diagnosing RA. The diagnostic accuracies of Fibß60-74 and Fibß36-52 were better than those of Vim60-75, Fibα36-50, and Fibα563-583, and all of them were better than that of Fibα621-635 and Fibα580-600.Key Points• Our study summarized the diagnostic accuracy of peptides derived from fibrinogen and vimentin and evaluated the diagnostic value of different peptides for RA patients.• Both fibrinogen and vimentin peptides have a high specificity but a relatively low sensitivity in diagnosing RA.

Diagnostic Accuracy of Anti-Carbamylated Protein Antibodies in Rheumatoid Arthritis: a Systematic Review and Meta-Analysis.

BACKGROUND: The purpose of this study was to estimate the diagnostic accuracy of anti-carbamylated protein (anti-CarP) antibodies in rheumatoid arthritis. METHODS: We searched the PubMed, EMBASE, Cochrane Library, Web of Science, and Scopus databases for studies published before January 1, 2019. Two investigators independently evaluated studies to determine their inclusion in the analysis, assess their quality, and extract the relevant data. The articles were assessed with the Quality Assessment of Diagnostic Accuracy Studies tool, and a bivariate mixed effects model was used to estimate the diagnostic indexes across studies. RESULTS: We included 16 published studies in this meta-analysis. The pooled sensitivity and specificity of anti-CarP were 43.1% and 94.4%, respectively. The area under the summary receiver operator characteristic curve was 0.55. The specificity estimates were highly heterogeneous, which could be partly explained by the higher specificity in the healthy control group (43.0%, 96.8%) than in the other disease group (43.4%, 89.8%). CONCLUSIONS: Anti-CarP antibodies have a relatively low sensitivity and high specificity for rheumatoid arthritis. However, the specificity was lower in the other disease subgroups than in the healthy controls.

Specific zinc finger-induced methylation of PD-L1 promoter inhibits its expression.

DNA methylation of promoter regions is often associated with epigenetic silencing of gene expression, and DNA methyltransferase (DNMTs) has been used to suppress gene expression. In order to explore the synergistic roles of two methyltransferase members Dnmt3a and Dnmt1, we constructed expression plasmid that could express a recombinant DNMTs consisting of the C-terminal domains of both Dnmt3a and Dnmt1 fused to a zinc finger domain which binds to the PD-L1 promoter of human prostate cancer cells (DU145). Programmed death ligand 1 (PD-L1, B7-H1, CD-274) is a transmembrane protein widely expressed on antigen-presenting and other immune cells. The interaction of PD-L1 with its receptor PD-1 is considered an 'immune checkpoint' for possible cancer therapy. DU145 cells treated with the Dnmt3aC-1C plasmid showed significantly reduced expression of PD-L1 as compared to Dnmt3aC or Dnmt1C alone. Our results show that the fusion of Dnmt1 improves the methylation activity of Dnmt3a and enhances its biological functions. This combinatorial strategy can be used to better control PD-L1 expression to support cytotoxic T lymphocytes (CTL) response against tumors.

Respiratory syncytial virus F and G protein core fragments fused to HBsAg-binding protein (SBP) induce a Th1-dominant immune response without vaccine-enhanced disease.

The induction of a dominant Th2-type response is the main cause of harmful inflammation in respiratory syncytial virus (RSV) vaccine trials. A balanced Th1 versus Th2 immune response is needed for a safe and effective RSV vaccine. In this study, we evaluated the potential of a recombinant protein SBP-FG as a vaccine candidate with the main focus on shifting the harmful Th2 response to a Th1 response. SBP-FG consists of epitopes from RSV fusion (F) and attachment (G) proteins conjugated to the N-terminus of HBsAg-binding protein (SBP). SBP-FG induced significantly stronger immune responses assessed at the level of total IgG, IgA and neutralizing antibodies as compared with formalin-inactivated RSV (FI-RSV) and live RSV. Analysis of IgG isotypes, lung cytokines and T helper cells showed that SBP-FG induced a dominant Th1-type response. Further, SBP-FG immunized mice showed significantly reduced lung eosinophilia, reduced viral multiplication in lungs after challenge infection and provided protection against RSV infection. These results suggest that SBP-FG can be developed into a safe and effective vaccine against RSV. However, more studies are required to further evaluate SBP-FG as a potent vaccine candidate against RSV.

Nasal immunization with RSV F and G protein fragments conjugated to an M cell-targeting ligand induces an enhanced immune response and protection against RSV infection.

Human respiratory syncytial virus (RSV) is a major paediatric health concern worldwide. The development of an effective and safe vaccine against RSV is urgently needed. As RSV infects via the mucosal surfaces, developing a nasal vaccine may offer protective benefits over alternative administration routes. In this study, we tested a recombinant protein FG-Gb1 as an intranasal vaccine candidate against RSV. FG-Gb1 consists of the core fragments of the RSV fusion (F) and attachment (G) proteins conjugated to an microfold (M) cell-specific ligand Gb-1. Intranasal immunization with FG-Gb1 induced efficient systemic and mucosal immune responses as measured by the level of antigen-specific antibodies, cytokine-secreting cells and antigen-specific lymphocyte proliferation after exposure to antigen. Moreover, intranasal immunization induced protective immunity against nasal challenge with RSV, which was confirmed by a lack of weight loss and by viral clearance after challenge. Collectively, we confirmed that a ligand capable of targeting the conjugated antigen to nasopharynx-associated lymphoid tissue (NALT) can be used as an effective nasal vaccine adjuvant to induce protective immunity against RSV infection. Moreover, FG-Gb1 may have promise as an RSV vaccine but requires further studies.

[Fermentation, purification and immunogenicity evaluation of hepatitis E virus-like particles expressed in Hansenula polymorpha].

To develop a new recombinant hepatitis E vaccine, we used Hansenula polymorpha expression system to express recombinant hepatitis E virus-like particles (HEV VLPs), to construct a recombinant engineered strain HP/HEV2.3. The fermentation conditions and purification process were studied next. The first working seed lots were fermented in liquid culture, and the fermentation products were collected, then crushed, clarified, purified by ultrafiltration, silica gel adsorbed and desorbed, concentrated by ultrafiltration, purified by liquid chromatography and sterilized by filtration. The purity reached 99% with a yield of 33%. Electron microscopy analysis revealed that both the purified recombinant HEV VLPs from HP/HEV2.3 and natural hepatitis E virus particles appear identical of being 32 nm. The resulting DNA sequence obtained from VLPs is identical to the published HEV sequence. The SDS-PAGE analysis has revealed that the protein molecular weight of the HEV VLPs is 56 kDa, and the expression product HEV VLPs were accumulated up to 26% of total cellular protein. The expression level is 1.0 g/L. Western blotting, enzyme-linked immunosorbent assay (ELISA) results of the protein and ED50 of the vaccine showed that the HEV VLPs have good antigenicity and immunogenicity. In summary, the recombinant HEV VLPs from Hansenula polymorpha can be used in the manufacture of a new genetically engineered vaccine against hepatitis E.

Systems Pharmacology-based strategy to screen new adjuvant for hepatitis B vaccine from Traditional Chinese Medicine Ophiocordyceps sinensis.

Adjuvants are common component for many vaccines but there are still few licensed for human use due to low efficiency or side effects. The present work adopted Systems Pharmacology analysis as a new strategy to screen adjuvants from traditional Chinese medicine. Ophiocordyceps sinensis has been used for many years in China and other Asian countries with many biological properties, but the pharmacological mechanism has not been fully elucidated. First in this study, 190 putative targets for 17 active compounds in Ophiocordyceps sinensis were retrieved and a systems pharmacology-based approach was applied to provide new insights into the pharmacological actions of the drug. Pathway enrichment analysis found that the targets participated in several immunological processes. Based on this, we selected cordycepin as a target compound to serve as an adjuvant of the hepatitis B vaccine because the existing vaccine often fails to induce an effective immune response in many subjects. Animal and cellular experiments finally validated that the new vaccine simultaneously improves the humoral and cellular immunity of BALB/c mice without side effects. All this results demonstrate that cordycepin could work as adjuvant to hepatitis b vaccine and systems-pharmacology analysis could be used as a new method to select adjuvants.

Phage Display-Derived Ligand for Mucosal Transcytotic Receptor GP-2 Promotes Antigen Delivery to M Cells and Induces Antigen-Specific Immune Response.

Successful oral immunization depends on efficient delivery of antigens (Ags) to the mucosal immune induction site. Glycoprotein-2 (GP-2) is an integral membrane protein that is expressed specifically on M cells within follicle-associated epithelium (FAE) and serves as transcytotic receptor for luminal Ags. In this study, we selected peptide ligands against recombinant human GP-2 by screening a phage display library and evaluated their interaction with GP-2 in vitro and ex vivo. Selected peptides were conjugated to the C-terminal of enhanced green fluorescence protein (EGFP) and evaluated for their ability to induce an immune response in mice. One of our selected peptides, Gb-1, showed high binding affinity to GP-2 and, when fused to EGFP, significantly increased the uptake of EGFP by M cells compared to EGFP alone. After oral administration, the Gb1-EGFP fusion induced efficient mucosal and systemic immune responses in mice measured at the level of antigen-specific serum and fecal antibodies, cytokine secretion, and lymphocyte proliferation. Furthermore, the IgG subclasses and cytokine secretion showed that ligand Gb-1 induced a Th2-type immune response. Collectively, our findings suggest that the ligand we selected through phage library screening is capable of targeting Ags to GP-2 on M cells and can be used as an oral vaccine adjuvant.

A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) Adjuvant for Hepatitis B Vaccine.

Although adjuvants are a common component of many vaccines, there are few adjuvants licensed for use in humans due to concerns about their toxic effects. There is a need to develop new and safe adjuvants, because some existing vaccines have low immunogenicity among certain patient groups. In this study, SBP, a hepatitis B surface antigen binding protein that was discovered through screening a human liver cDNA expression library, was introduced into hepatitis B vaccine. A good laboratory practice, non-clinical safety evaluation was performed to identify the side effects of both SBP and SBP-adjuvanted hepatitis B vaccine. The results indicate that SBP could enhance the HBsAg-specific immune response, thus increasing the protection provided by the hepatitis B vaccine. The safety data obtained here warrant further investigation of SBP as a vaccine adjuvant.

Rabies virus lipopeptide conjugated to a TLR7 agonist improves the magnitude and quality of the Th1-biased humoral immune response in mice.

In this study, we conjugated the rabies-derived lipopeptide CE536 to a TLR7 agonist, imiquimod, and evaluated its adjuvanticity. The synthetic construct (Lipo-I) targeted to TLR7, induced dendritic cell phenotypic maturation and production of both type I interferon and pro-inflammatory cytokines more efficiently than unconjugated TLR7 ligands or lipopeptide alone. The immunostimulatory effects of the conjugate were apparently the result of IκBα degradation and sustained p38 and JNK phosphorylation. The analysis of IgG isotypes and T cell differentiation showed that IgG2a dominant Th1-biased humoral and CD8(+) IFN-γ T cell responses were induced by Lipo-I. Lipo-I could facilitate the rabies vaccine to induce the production of an earlier and more vigorous rabies virus neutralizing antibody. In the post-exposure test, the Lipo-I adjuvanted vaccine provided a 73.3% survival rate, while the traditional vaccine bestowed only a 26.7% survival. Therefore, Lipo-I is a promising adjuvant for the development of more effective rabies vaccines.

A novel rabies virus lipopeptide provides a better protection by improving the magnitude of DCs activation and T cell responses.

Besides rabies virus neutralizing antibody, non-neutralizing antibody to internal vital proteins, interferon, and possibly cell-mediated immunity also have a critical role in preventing the infection by rabies virus (RV). We identified novel CTL and Th epitopes which could induce lymphocyte proliferation and IFN-γ, IL-4 production, and designed linear and branched lipopeptides with these selected CTL and Th epitopes. Compared to linear construct, branched lipopeptides, especially Lipo C, stimulate stronger phenotypic and functional maturation of DCs, as well as more efficient CD8(+) T cell responses, evaluating by using FACS, G333-341 tetramer staining and specific CTL assay. Lipo C could also assist rabies vaccine to induce an instant rabies virus neutralizing antibody production, and better protection against rabies virus challenge at early stage. These data reveal that Lipo C could be a promising component for developing novel rabies vaccines.

eEF1A1 binds and enriches protoporphyrin IX in cancer cells in 5-aminolevulinic acid based photodynamic therapy.

Photodynamic therapy (PDT) with protoporphyrin IX (PpIX), which is endogenously derived from 5-aminolevulinic acid (5-ALA) or its derivatives, is a promising modality for the treatment of both pre-malignant and malignant lesions. However, the mechanisms of how ALA-induced PpIX selectively accumulated in the tumors are not fully elucidated. Here we discovered that eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) interacted with PpIX (with an affinity constant of 2.96 × 10(6) M(-1)). Microscopy imaging showed that ALA-induced PpIX was co-localized with eEF1A1 in cancer cells. eEF1A1 was found to enrich ALA-induced PpIX in cells by competitively blocking the downstream bioavailability of PpIX. Taken together, our study discovered eEF1A1 as a novel photosensitizer binding protein, which may play an essential role in the enrichment of ALA-induced PpIX in cancer cells during PDT. These suggested eEF1A1 as a molecular marker to predict the selectivity and efficiency of 5-ALA based PDT in cancer therapy.

Single primer-mediated circular polymerase chain reaction for hairpin DNA cloning and plasmid editing.

We developed and validated a universal polymerase chain reaction (PCR) method, single primer circular (SPC)-PCR, using single primer to simultaneously insert and amplify a short hairpin sequence into a vector with a high success rate. In this method, the hairpin structure is divided into two parts and fused into a vector by PCR. Then, a single primer is used to cyclize the chimera into a mature short hairpin RNA (shRNA) expression vector. It is not biased by loop length or palindromic structures. Six hairpin DNAs with short 4-nucleotide loops were successfully cloned. Moreover, SPC-PCR was also applied to plasmid editing within 3 h with a success rate higher than 95%.