The aim is to identify the possible performance of human lysozyme-like protein 6 (LYZL6) in fertilization and to characterize its physiological properties. Immunofluorescent staining with a specific antiserum localized the LYZL6 protein on the postacrosomal membrane of mature spermatozoa, which was secreted by testis and epididymis as demonstrated by the reverse transcription polymerase chain reaction (RT-PCR). No significant decrease of LYZL6 after capacitation was observed by Western blot analysis. Immunoneutralization of LYZL6 showed no effect on the binding of spermatozoa to the hemizona, but significantly decreased the numbers of human spermatozoa fused to zona-free hamster eggs in a dose-dependent manner. The Pichia expression system was utilized to produce recombinant LYZL6 (rLYZL6). After induction with methanol, rLYZL6 was purified from the fermentation supernatant by chitin affinity in combination with gel-filtration chromatography. In vitro assays indicated that rLYZL62 possessed no hyaluronan-binding ability, hyaluronidase activity and free radical scavenging activity, but peptidoglycan-binding ability and isopeptidase activity. In conclusion, LYZL6, a human sperm-related protein is reported, not only plays a role in sperm-egg fusion but also has peptidoglycan-binding ability and isopeptidase activity, suggesting it might contribute to diverse sperm functions.
To explore biological function of Herpud1 in kidney development, overexpression and knockdown vector of Herpud1 gene were transfected into MK3 cells. And then RT-PCR and Western blot were used to detect expression of epithelial mesenchymal transition (EMT) marker genes E-cadherin (epithelial cells marker), Vimentin and Snail (mesenchymal cell marker) and endoplasmic reticulum (ER) stress marker genes (GRP78 and eIF2α). And cell proliferation and migration were detected by EDU cell proliferation experiment and wound healing assay. Our results showed in experimental group with Herpud1 overexpression, E-cadherin was decreased, and expression of Vimentin and Snail was increased at mRNA and protein levels compared with control group. And ER stress markers GRP78 and eIF2α were enhanced at protein levels in MK3 cells with Herpud1 overexpression. In addition, Herpud1 promoted cell migration and inhibited cell proliferation. In Herpud1 knockdown cells, expression of E-cadherin was enhanced at mRNA and protein levels, and expression of Vimentin, Snail, GRP78 and eIF2α is reduced. At the same time, Herpud1 knockdown inhibited cell migration and enhanced ability of cell proliferation. These results demonstrate Herpud1 can promote EMT of MK3 cells, cell migration and inhibit cell proliferation. The mechanism may be associate with ER stress.
To prokaryotically clone and express phosphoglycerate kinase(PGK) from Streptococcus suis serotype 2 and determine enzymatic properties of recombination protein.
pgk gene was amplified from the 05ZYH33 genome DNA by PCR and inserted into expression vector pET28a by double digestion. The combined prokaryotic expression plasmid pET28a:pgk was subsequently transformed into E.coli BL21 and induced by IPTG. The induction result was identified by SDS-PAGE and LC-MS/MS, the recombination PGK was then purified by Ni affinity chromatography and used for enzymatic activity measurement.
PGK protein had a high and soluble expression and displayed a molecular weight about 43kDa in E.coli BL21. The enzymatic activity was then measured by using purified PGK. The enzymatic activity of recombination PGK protein was 75U/ml. The optimum temperature and pH was 25℃ and 7.5, respectively. Kinetic analyses with respect to 3-PGA as substrate gave a Km of 1.744mmol/L and ATP as substrate gave a Km of 2.266mmol/L.
The pgk gene has been successfully expressed by prokaryotic expression system and the purified recombinant PGK protein had a similar enzymatic activity other protein enzymes and showed the best enzymatic activity in optimum condition.
Directional regulation effect of Eucalyptus urophylla COMT gene and CCoAOMT gene on lignin monomer synthesis were studied by tobacco transformation system. Transgenic plants C-S(contain sense EuCOMT fragment), CR(contain full-length RNAi fragment of EuCCoAOMT), C-CR(contain EuCOM and EuCCoAOMT dual-gene) were obtained, and the expression of sense EuCOMT fragment was detected in transgenic tobacco, while the RNAi fragment of EuCCoAOMT caused strong inhibition on tobacco CCoAOMT gene. The growth morphology, lignin content, fiber content and anatomical structure of transgenic tobacco were not significantly different from those of wild type. The results of lignin monomer detection showed the G lignin content increased 17.72% and S/G ratio decreased 17.99% in transgenic plant C-S, meanwhile the S/G ratio increased 61.62% in CR. In C-CR, G lignin content decreased 57.38%, and S/G ratio increased 114.94%. The result showed that the inhibition of CCoAOMT had a significant inhibitory effect on lignin synthesis in G, and the dual-gene transformation of EuCOMT and EuCCoAOMT showing the best directional regulation effect.
Leaf senescence is the last phase of plant development, environmental factors such as dark, drought, extreme temperature can promote leaf senescence, which may influence the plant growth and crop yield. Basic/helix-loop-helix(bHLH) transcription factors (TFs)belong to a superfamily of transcriptional regulators, it is named bHLH for its highly conserved bHLH domain, and widely exists in the eukaryotes. A candidate bHLH encoding gene, CibHLH027, was screened from the transcriptome library of Caragana intermedia. Compared with wild type, CibHLH027-OE promoted leaf senescence under both light and darkness, with reduced chlorophyll content, increased ion leakage and cell death under darkness, indicates that CibHLH027 is involved in leaf senescence.
Cadmium is an extremely toxic pullutant. The characterists of having long half-life and resistance to environmental degradation make it easy to accumulate in human organs and then cause irreversible damage. Blocking cadmium uptake via the gastrointestinal tract is an important prevention strategy.
the recombinant Lactococcus lactis strain MG1363/pM-GSMT were constructed, which expressed fusion protein glutathione S-transferase(GST)-small molecule ubiquitin-like modifier protein (SUMO)-metallothionein-I (GST-SUMO-MT) and using α- galactosidase as marker. The ability of binding cadmium or zinc ions was detected by the Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). For the animal experiments, the male Sprague-Dawley rats were given a daily oral administration of cadmium chloride solution [5mg/(kg·d)] and MG1363/pM-GSMT with different dosages respectively. This experiment was performed for 8 weeks and the tissues (liver, kidney, brain and testis) were collected. The accumulation of cadmium in tissues was determined by atomic absorption spectrometric analysis. The concentrations of alanine aminotransferase (ALT) and blood uria nitrogen (BUN) were analyzed by a full automated hematology analyzer. The pathological changes of each tissue treated by cadmium with/without MG1363/pM-GSMT were compared and analyzed using hematoxylin - eosin staining.
The recombinant strain MG1363/pM-GSMT with α-galactosidase resistant marker was constructed, which binding capacity of cadmium and zinc ions were 3.52 and 1.60 fold higher than that of control strain MG1363/pMG36e. After daily CdCl2 treatment for 8 weeks, the results showed that the cadmium concentration increased significantly in the kidney, liver, brain and testis of experimental group compared with the normal group. The accumulated cadmium impaired the functions of liver and kidney. Oral administration of MG1363/pM-GSMT to SD rats that were also treated with cadmium chloride daily significantly blocked the cadmium uptake via gastrointestinal tract and further decreased the impairments of main tissues in morphology and functions.
A potential biomaterial and application for preventing cadmium poisoning via the digestive tract.
SUMO protease (Ulp1) is an enzyme that cleaves small molecule ubiquitin-modified (SUMO) fusion protein into natural N-terminal protein,which has high efficiency and specificity.While the existing commercially available SUMO protease Ulp1 is expensive and complicated on operational level,limiting the wide application of SUMO fusion system. ulp1 gene (Leu403-Lys621) was synthesized,with the poly-histidine tag (His6) added to both the N-terminal and C-terminus of the SUMO protease(Ulp1).The recombinant expression vector psvT7-ulp1 was constructed before the recombinant plasmids was transformed into Escherichia coli BL21(DE3) and BL21 trxB(DE3) separately.The optimal expression conditions were as follows:Escherichia coli BL21 (DE3) was used as the expression host,and IPTG was added at 7h after the transfer.The final concentration of IPTG was 0.1mmol/L and the induction time was 16h.The final recombinant protein Ulp1 was 190mg/L accounted for 34.5% of the total protein in bacteria.Purified SUMO could be obtained through one-step Ni-NTA and the purity was higher than 95%.The enzymatic activity was 5.19U/μl with a specific enzyme activity of 5.23×10 4U/mg,which was 1.87 folds of the previously reported,according to enzyme kinetic analysis,the apparent Km of Ulp1 was 0.359g/L,and Vm was 5.10μg/(ml·min).The SUMO fusion expression system was then used to express the soluble SUMO-scFv fusion protein,with the expressed Ulp1 used for digestion and purification.The purity of scFv was higher than 90% and N-terminal natural scFv was obtained. A method was presented for preparation of Ulp1 with high enzyme activity and purity.The SUMO fusion expression system was successfully applied to the expression and purification of scFv with a simplified operation steps,which significantly improved the status of scFv soluble expression in Escherichia coli.
To increase the expression level of soluble tumor necrosis factor type-I receptor(sTNFαRI)in E.coli BL21(DE3)by optimizing the secondary structure of PET11b-sTNFaRI translation initiation region(TIR).
The free energy and nucleotide position entroy of the secondary structure of the translational initiation region was analyzed as the first step, and the primers were designed to mutate the codons of TIR of the PET11b-sTNFαRI in order to exposure of ribosome binding site and start codon to the outside of hairpin structure, in addition to mutating the pET11b ribosome binding site from GAAGGAGA to GAAGAA in order to facilitate assembly of the translational complex and initiation of translation. The optimized sequence of 5' terminal TIR was cloned into PET11b vector and transformed into E.coli BL21(DE3). The positive transformants were induced by IPTG and analyzed by SDS-PAGE and Western blot.
SDS-PAGE and Western blot analysis showed that the expression of Recombinant sTNFαRI was increased by 50%~60%, after optimizing the secondary structure of 5' terminal TIR of PET11b-sTNFαRI.
The optimization of the secondary structure of the translation initiation region(TIR)mRNA of recombinant vector can effectively increase the expression level of the target protein, which is of great value for further industrialized production.
In medicine, the ability of the nervous system to repair nerve damage is often limited. In recent years, it has been found that adipose derived stem cells (ADSCs) have repair effects on various types of nerve injury and can be the seed cells for nerve injury repair. Not only being as stem cells, ADSCs also have some exclusive advantages. ADSCs belong to adult cells, which derived from the mesoderm, with multi-lineage differentiation potential, low immunogenicity, easy to gain, low risk after transplantation, thus make them become excellent immunogenicity, easy to gain, low risk after transplantation, thus make them become excellent seed cells for the nerve repair.The characteristics of adipose derived stem cells and their research progress and existing problems on nerve injury repair were reviewed.
Nervous system diseases occur in the central, peripheral, autonomic nervous system with sensory, motor, consciousness, autonomic nervous dysfunction as the main manifestation of the disease, is currently one of the most harmful to human diseases. With the development of stem cell biology, human beings have made remarkable progress in inducing neural differentiation of stem cells and may solve the key problems of nerve repair and regeneration. Small molecule compounds are increasingly used to intervene and study the biological behaviors of stem cells such as proliferation, differentiation and reprogramming because of their obvious advantages in convenience, controllability and functional diversity.The progress of neural cell differentiation induced by small molecule compounds in stem cells is reviewed.
The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) is an adaptive immune system. CRISPR/Cas9 technology can be used to edit single or multiple genes in a wide variety of cell types and organisms in vitro and in vivo. In recent years, some of the gene editing techniques, such as Zinc finger nuclease and transcriptional activator-like effector nuclease, have brought a lot of convenience to gene editing. But CRISPR/Cas9 system with its unique features has brought a revolutionary change to the field of gene editing. CRISPR/Cas9 technology has been widely used in the fields of biological research, the treatment of gene-related diseases, and the research of disease in gene level. The research progress of CRISPR/Cas9 delivery system and its application in genetic diseases are reviewed, and the pros and cons of lentivirus and adeno-associated virus vectors in the delivery of gene editing elements are discussed,and the problems that still exist in the CRISPR/Cas9 system are explored.
Lysyl endopeptidase is an important enzyme as a tool for biotechnological purposes and industrial production. However, its applications are obstructed by its high costs of production, due to the current lysyl endopeptidase products are extracted from the native bacteria with very low yields. Nevertheless, the recombinant expression system have solved this problem perfectly. A review of recent progress in lysyl endopeptidase for the first time was presented. The origins, structure and function of lysyl endopeptidase were mainly focused on. Furthermore, the successful expression of recombinant lysyl endopeptidase were aslo summarized and analyzed. In addition, the potential perspective of lysyl endopeptidase for future research were further discussed.
Streptococcus suis is an infectious Gram-positive bacterium. Streptococcus suis serotype 2 (S. suis 2, SS2) is an important zoonotic pathogen that severely affects the swine breeding industry and causes human mortality rates of between 5% and 20%. Its virulence factors play an important role in the pathogenesis. In recent years, there were many new advances in the virulence factors of SS2. in the pathogenic mechanism, and there were either new knowledge for effective prevention and control of the disease. The recent progress in the virulence-related factors of SS2, e.g. proteins, enzymes, the virulence factor gene expression two-component system and type IV secretion system which interaction to host immune system were summarized. There were valuable references for the treatment of SS2 and vaccine development.
Ascorbic acid (AsA) plays important roles in the growing development and stress resistance in plant. The pathways of AsA metabolism is revgiewed, and regulation mechanism of various genes on AsA metabolism by gene transcription, translation and transformation in model and horticultural plant are focused on. The reviews may provide insight for future study on regulation mechanism of AsA metabolism in plants, focusing specifically on the horticultural plant.
