Constructing DNA logic circuits based on the toehold preemption mechanism.

Strand displacement technology and ribozyme digestion technology have enriched the intelligent toolbox of molecular computing and provided more methods for the construction of DNA logic circuits. In recent years, DNA logic circuits have developed rapidly, and their scalability and accuracy in molecular computing and information processing have been fully demonstrated. However, existing DNA logic circuits still have some problems such as high complexity of DNA strands (number of DNA strands) hindering the expansion of practical computing tasks. In view of the above problems, we presented a toehold preemption mechanism and applied it to construct DNA logic circuits using E6-type DNAzymes, such as half adder circuit, half subtractor circuit, and 4-bit square root logic circuit. Different from the dual-track logic expressions, all the signals in the circuits of this study were monorail which substantially reduced the number of DNA strands in the DNA logic circuits. The presented preemption mechanism provides a way to simplify the implementation of large and complex DNA integrated circuits.

Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice.

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP) is the major protective antigen of EBOV, and can generate virus-like particles (VLPs) by co-expression with matrix protein (VP40). In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV) replicon vector DREP to express EBOV GP and matrix viral protein (VP40). EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40). Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention.

[Preparation and antigenicity analysis of recombinant aegyptin-like protein of Aedes albopictus].

OBJECTIVE: To clone and express the aegyptin-like protein (alALP) encoding gene from Aedes albopictus salivary gland, and analyze its antigenicity. METHODS: The homology, secondary structure and antigen peptides of alALP and aegyptin protein (GenBank No. ABF18122.1) was analyzed by bioinformatics software tools. Total RNA was extracted from Ae. albopictus salivary gland. The coding region of alALP (GenBank No. AY826121) was amplified by PCR. RT-PCR product was digested with restriction enzyme and ligated into a pGEX-6P-1 vector. The recombinant pGEX-6P-1-alALP plasmid was transformed into E. coli BL21 and induced by IPTG. The recombinant soluble GST-alALP fusion protein was purified with Glutathione Sepharose 4B. The expression product was analyzed by SDS-PAGE and Western blotting. Mice were immunized each with 60 microg purified GST-alALP at every 2 weeks for 3 times, and mouse anti-GST-alALP serum was prepared. Western blotting assay with mice anti-GST-alALP serum and serum of mice exposed to Ae. albopictus bites was used to analyze its antigenicity. RESULTS: Bioinformatics prediction results showed that alALP and aegyptin had 65.58% homology with a similar secondary structure, and a conservative polypeptide. The product of RT-PCR was 762 bp. The recombinant plasmid pGEX-6P-1-alALP was confirmed by double restriction enzyme digestion, PCR and sequencing. SDS-PAGE and Western blotting analysis showed that the bacteria containing recombinant plasmid pGEX-6p-1-alALP expressed a soluble recombinant fusion protein (M(r) 56 000) after being induced with IPTG. Western blotting analysis revealed that GST-alALP protein was recognized by mouse anti-GST-alALP serum and serum of mice ex- posed to Ae. albopictus bites. CONCLUSION: Mature peptide gene of alALP can be expressed in prokaryotic expression system, and the recombinant protein shows antigenicity.

Identification and characterization of the expression profile of microRNAs in Anopheles anthropophagus.

BACKGROUND: Anopheles anthropophagus, one of the most important mosquito-borne disease vectors in Asia, mainly takes blood meals from humans and transmits both malaria and filariae. MicroRNAs (miRNAs) are small non-coding RNAs, and play a critical role in many cellular processes, including development, differentiation, apoptosis and innate immunity. METHODS: We investigated the global miRNA expression profile of male and female adults of A. anthropophagus using illumina Hiseq2000 sequencing combined with Northern blot. RESULTS: By using the miRNAs of the closely-related species Anopheles gambiae and Aedes aegypti as reference, we obtained 102 miRNAs candidates out of 12.43 million raw sequencing reads for male and 16.51 million reads for female, with 81 of them found as known miRNAs in An. gambiae and/or Ae. aegypti, and the remaining 21 miRNAs were considered as novel. By analyzing the revised read count of miRNAs in male and female, 29 known miRNAs show sexual difference expression: >2-fold in the read count of the same miRNAs in male and female. Especially for miR-989, which is highly expressed in the female mosquitoes, but shows almost no detected expression in male mosquitoes, indicating that miR-989 may be involved in the physiological activity of female mosquito adults. The expression of four miRNAs in different growth stages of mosquito were further identified by Northern blot. Several miRNAs show the stage-specific expression, of which miR-2943 only expressed in the egg stage, suggesting that miR-2943 may be associated with the development of mosquito eggs. CONCLUSIONS: The present study represents the first global characterization of An. anthropophagus miRNAs in sexual differences and stage-specific functions. A better understanding of the functions of these miRNAs will offer new insights in mosquito biology and has implications for the effective control of mosquito-borne infectious diseases.