Abstract:Functional genetic screening as an important method for exploring biological processes, diseases development research and functional annotation of genetic elements, has been widely used in pharmaceutical research, new therapeutic targets identifying and screening, and tumor resistance. CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9) is the newest tool in the geneticist’s toolbox, allowing researchers to edit genome with unprecedented ease, accuracy and high-throughput. CRISPR-Cas9 system provides a high-throughput, practical and efficient tool for the discovery of functionally important genes responsible for certain phenotypes. In this review, we summarize the characterization of CRISPR/Cas9 system and applications of this new genetic toolkit in functional genetic screening.

Abstract:Plant type Ⅲ polyketide synthases (PKSs), the pivotal enzymes in the biosynthesis of polyketides, produce backbones of many structurally diverse and functionally different polyketides. So far, a variety of functionally diverse plant type Ⅲ PKSs have been cloned and identified from plant origin. Site-directed mutagenesis is a useful technique to study the complex relationship between protein structure and function. This review summarized advances in the structure-function relation of plant type Ⅲ polyketide synthases by site-directed mutagenesis in recent years, including the modification of the amino acid residues influencing enzyme architectures (such as controlling the specificity of starter substrates, the number of condensation reactions, and the cyclization reactions of the intermediate product). This review provides information to study the structure-function relation of plant type Ⅲ polyketide synthases.

Abstract:Zearalenone (ZEN) and its derivatives are non-steroidal estrogenic mycotoxins mainly produced by Fusarium species. They are widely distributed in grain feeds originated from maize, barley, wheat and sorghum, causing serious harm to animal and human health. Currently, there is a pressing need of an efficient technology for ZEN degradation and detoxification. Because traditional physical and chemical methods could not effectively detoxify ZEN in grains, and might also affect the grain nutrients and food taste, and even result in secondary pollution, the biological technologies are developed to detoxify ZEN and its derivatives. In this paper, we reviewed the structure of ZEN and its derivatives, the fungi and bacteria species with ability of degradation of ZEN. In addition, the characterization, protein sequences and conformation of currently identified ZEN degrading enzymes, the only solved ZHD structure from Clonostachys rose were analyzed and compared, and the enzymes heterologous expression and application were also reviewed. This review will provide reference for reducing the cost of ZEN degrading enzymes by biological technologies such as enzyme engineering and fermentation engineering.

Abstract:Poly (3-hydroxypropionate) (P3HP), a new member of thermoplastic of family polyhydroxyalkanoates (PHAs), has excellent characteristics of biodegradability and biocompatibility. By now no reports can be found about wild-type bacteria that naturally synthesize P3HP, so the main way to produce P3HP is chemical and biological methods. Chemical method by adding high cost 3-HP monomers or their structural analogs as precursors, has the drawbacks of toxicity, low effectiveness and high cost. Biological method using engineered strain may utilize inexpensive and renewable carbon source to produce P3HP and has gradually become more and more popular. We systematically review here the biosynthesis of P3HP research progress. The advantages and disadvantages of biosynthesis pathways of glycerol pathway, malonyl-CoA pathway and β-alanine pathway were analyzed.

Abstract:Chlorinated aliphatic hydrocarbons (CAHs) with characteristics of high toxicity, biological accumulation and recalcitrance to degradation as well as carcinogenicity, teratogenesis and mutagenicity, are seriously harmful to human health and ecological environment. CAHs degradation depends on biotic and abiotic responses that exist diversified interactive effects, so it is important to clarify the mechanism of CAHs degradation via biotic and abiotic mutual promoting to significantly enhance the CAHs-contaminated site restoration. In this work, a series of pathways for CAHs degradation was first introduced and summarized as three means on reductive dechlorination, aerobic cometabolism and direct oxidation, and biotic and abiotic typical factors affecting CAHs degradation were concluded from these. Then, mechanisms of induced degradation and synergistic degradation were indicated from the perspective of mutual promoting degradation both with biotic and abiotic responses, and furthermore, the application and technical limitations of CAHs degradation enhanced via biotic and abiotic mutual promoting were reviewed and analyzed. Finally, the development of CAHs degradation technology in future was prospected.

Abstract:Exponential growth of the mass spectrometry (MS) data is exhibited when the mass spectrometry-based proteomics has been developing rapidly. It is a great challenge to develop some quick, accurate and repeatable methods to identify peptides and proteins. Nowadays, the spectral library searching has become a mature strategy for tandem mass spectra based proteins identification in proteomics, which searches the experiment spectra against a collection of confidently identified MS/MS spectra that have been observed previously, and fully utilizes the abundance in the spectrum, peaks from non-canonical fragment ions, and other features. This review provides an overview of the implement of spectral library search strategy, and two key steps, spectral library construction and spectral library searching comprehensively, and discusses the progress and challenge of the library search strategy.

Abstract:The high prevalence of influenza A virus is identified in Hunan Province because of the high density of poultry farms. To survey the variations of H9N2 subtype avian influenza virus in Hunan province, we analyzed HA and NA genes of 10 virus strains isolated from different areas of Hunan Province. All these strains belong to the Eurasian lineage, Y280-like sub-lineage. The cleavage sites in their HA genes were all RSSR↓GLT, corresponding to the feature of low pathogenic AIV. All strains had an L (Leu) at the site 234 in the HA genes, indicating the ability of binding with the SAα-2,6 receptor. NA gene stalk deletions at aa 63–65 were also detected from all the isolates, indicating a possibility of increased virus replication in mammals. Our findings suggest that more attention should be paid to the surveillance of H9N2 influenza virus and its direction of reassortment.

Abstract:The emission of hydrogen sulfide in the waste gas from slaughter plant, fishmeal feed processing and some other food industrial processing could cause serious air pollution to the surrounding environment. The purpose of this study was to screen heterotrophic bacterium strains for the removal of hydrogen sulfide odor. One heterotrophic bacterial mutant ZJNB-B3 was derived from the sulfide degrader?Bacillus cereus XJ-2 and its sulfide removal efficiency was 97%. Based on the morphology studies, biochemical tests and 16S rRNA gene analysis, the strain was identified as Bacillus cereus ZJNB-B3. The NCBI GenBank accession number is MF679650. Batch tests showed that the strain tolerated up to 300 mg/L of toxic S2– concentration. Response surface methodology was applied to optimize the conditions of degradation of sulfide. The optimal parameters were as follows: initial sulfide concentration 211.8 mg/L, initial pH 6.72, inoculum volume 5.04%, and incubation temperature 30 ℃. The accumulated sulfate concentration was 63.8 mg/L and the sulfide removal efficiency was 97.3% after 48 h incubation. No sulfuric acid was generated during sulfide oxidation by the strain. Sulfide could be removed effectively by this strain under mild pH conditions. The results suggested that the strain may have great industrial application potential. This study provides the fundamentals for the removal of hydrogen sulfide gas.

Abstract:CRM197 (cross-reacting material 197), a non-toxic mutant of diphtheria toxin, has wide application potential in biopharmaceuticals. However, it is difficult to express CRM197 in bacteria other than Corynebacterium diphtheriae. Here we proposed a new alternative method to produce soluble CRM197 without label in Escherichia coli. In particular, a synthetic gene coding for CRM197, optimized for E. coli codon usage, was cloned in the pET32a (+) vector. Accordingly, the over-expression of the protein was simply induced with IPTG in E. coli BL21 (DE3). The target protein was soluble and accounted for about 40% of the total protein in the supernatant. Following an ultrasonic cytolysis step, the recombinant protein was purified by anion exchange, affinity and desalting chromatography and the purity of the final preparation reached 95%. Cytotoxicity tests showed that the IC50 value of CRM197 was 2.1×107 times the IC50 value of diphtheria toxin, and 9.6 times the IC50?value of diphtheria toxoid, telling that the target protein is safe and non-toxic. Subsequently, we found that both the high dose (20 μg) and the low dose (2 μg) of CRM197 were equally efficient in inducing an immune response against diphtheria?toxiod in mice, and the antibodies titer of mice after three immunizations with low dose could reach 1:409 600. In conclusion, our findings provide a highly efficient strategy for the rapid production and purification of unlabeled and soluble recombinant CRM197 in E. coli, with good immunogenicity and safety.

Abstract:We used the antibody grafting technology to prepare anti-hCG single-domain antibodies on the basis of antigen-binding peptide to simplify the single-domain antibody preparation process and improving the biochemical stability of peptide. By using a universal single-domain antibody backbone (cAbBCII10), CDR1 or CDR3 was replaced by the hCG-binding peptide, and the grafted antibody gene sequences were synthesized and cloned into the prokaryotic expression vector pET30a(+) in fusion with a C-terminal sfGFP gene, i.e. pET30a-(His6)-cAbBCII10-CDR1/hCGBP1-sfGFP and pET30a-(His6)-cAbBCII10- CDR3/hCGBP3-sfGFP. The recombinant plasmids were transformed into E. coli BL21(DE3), and the fusion proteins were induced by IPTG.?Highly soluble recombinant fusion proteins were obtained and purified by Ni-NTA affinity column. SDS-PAGE confirmed the purified protein as the target protein. The antigen-antibody binding assay showed that?both the CDR1 and CDR3 grafted antibodies have hCG-binding activities. While the titers of the two grafted antibodies were similar, the binding affinity of CDR3 grafted antibody was higher than that of CDR1 grafted protein (about 2–3 times). The grafted antibodies retained the relatively high biochemical stability of the single-domain antibody backbone and were relatively thermostable and alkaline tolerant. The obtained antibodies also had a relatively high antigen-binding specificity to hCG. This study provided a reliable experimental basis for further optimization of anti-hCG single domain antibody by antibody grafting technology using antigen-binding peptide.

Abstract:Biomarkers are the detectable changes associated with physiological or pathological changes. Urine as excreta of the body, without the mechanisms to maintain a homeostatic internal environment, can reflect a variety of changes in the body. Using animal models can simulate human disease processes, monitor disease changes, and provide clues to early diagnosis. Rats as commonly used model animals are not the dominant models for all disease, thus comparing the urinary proteins of rats with other animals to provide clues to the selection of other animal dominant models. In this study, urinary proteins were digested and profiled by liquid chromatography and tandem mass spectrometry (LC-MS/MS). The urinary proteins of rats, guinea pigs and golden hamsters were compared. The results showed that the number of urine proteins in the three different animals was different, and also different in every system of the body. This provides a basis for selecting the best animal models for different diseases.

Abstract:We constructed the CAP2NC prokaryotic expression vector of HIV-1 NL4-3 strain and obtained relatively pure CAP2NC protein by optimizing its purification conditions to explore its in vitro self-assembly conditions. Primers were designed according to the CAP2NC DNA sequence of HIV-1 NL4-3 strain. The target gene was amplified by PCR and cloned into prokaryotic expression vector pTO-T7. Then the recombinant strain was transformed into Escherichia coli BL21 (DE3). IPTG induced protein expression, then the protein was purified by hydrophobic chromatography. SDS-PAGE and Western blotting were performed to analyze the target protein, and the biological activity of the antigen was identified through ELISA. The self-assembly of CAP2NC protein was analyzed by transmission electron microscopy and gel filtration chromatography. The protein had good reaction with the specific antibodies of p24 and formed different structures in various conditions. When 10% yeast RNA was added to the protein complex, the recombinant protein only formed into a tubular structure, which was similar to the self-assembled structure of the HIV-1 virus capsid. The results showed that the HIV-1 CAP2NC protein had in vitro self-assembly activity, and the RNA affected the structure of CAP2NC protein assembly. The protein can be used as a simple and effective molecular model to study its structure, and then it can provide a reference for the study of HIV immature virus particles.

Abstract:Neuropeptides play an important role in the physiological functions of the human body. The physiological activities such as pain, sleep, mood, learning and memory are affected by neuropeptides. Neuropeptides mainly exist in the nerve tissue of the body, and a small amount of them are distributed in body fluid and organs. At present, analysis of large-scale identification of neuropeptides in whole brain tissue is still challenging. Therefore, high-throughput detection of these neuropeptides is greatly significant to understand the composition and function of neuropeptides. In this study, 1 830 endogenous peptides and 99 novel putative neuropeptides were identified by extraction of endogenous peptides from whole brain tissue of mice by liquid phase tandem mass spectrometry (LC-MS / MS). The identification of these endogenous peptides provides not only a reference value in the treatment and mechanism studies of diseases and the development of drugs, but also the basis for the study of a new neuropeptides and their functions.

Abstract:Gene modification is an important technique to understand gene function. We firstly constructed Δhfq::Spe and Δrne-710::Spe mutant strains of Escherichia coli MG1655. The fragment lacking of hfq and rne-710 was ligated to the auxiliary plasmid and separately replace the spectinomycin box by homologous recombinase system to obtain the Δhfq and Δrne-710 mutant strains. The combination of two-plasmid scarless genetic modification and fusion PCR led to a new way for the long DNA fragment gene deletions.

Abstract:IFN-λ1 is a member of a new family of interferons called type Ⅲ IFNs with similar functions to type ⅠIFNs. Previously we obtained recombinant soluble human rhIFN-λ1 from Pichia pastoris. However, the hyper-glycosylation from P.?pastoris brings immunogenicity and low purification recovery rate. To overcome this disadvantage, in this study, we constructed an rhIFN-λ1 mutant (rhIFN-λ1-Nm) devoid of the potential N-glycosylation sites by site-directed mutagenesis. rhIFN-λ1-Nm was successfully expressed and secreted extracellularly in P. pastoris (GS115) using methanol inducible AOX1 promoter with α-mating factor signal sequence. rhIFN-λ1-Nm was purified and characterized. There was no significant difference between rhIFN-λ1-Nm and rhIFN-λ1 in structure and bioactivity. The molecular weight was low after N-glycosylation mutation whereas the glycosylation was much lower. The mutational rhIFN-λ1 (rhIFN-λ1-Nm) could legitimately be developed as substitutes for rhIFN-λ1, and thus it may be developed into a more promising therapeutic reagent in the future.