Objective: Progesterone (P4), as a reproductive hormone, changes regularly in the estrus cycle of mammals, and plays an essential role in the establishment and maintenance of pregnancy in cattle. This study through monitoring serum progesterone concentration in cattle, in addition to the routine manual estrus observation, screens for embryo transfer (ET) recipient cattle, as well as for the monitoring of reproductive status during the whole pregnancy of cattle upon Somatic Cell Nuclear Transfer (SCNT) experiments. Methods: Through the study of serum progesterone levels of natural-breeding cattle at different reproductive stages, the reference indexes of progesterone concentration at early estrus and during pregnancy were established. Based on these indexes, the recipient cattle suitable for embryo transfer were screened and selected by complementing manual estrus observation. At the same time, reconstructed blastocyst from SCNT cultured in vitro for seven days was transferred into the uterus of selected estrus-synchronized recipient cattle. The pregnancy state was monitored with serum progesterone level. Experimental results: (1) When using serum progesterone level to screen for SCNT embryo transfer recipient cattle, i.e. progesterone concentration at 0d (≤0.64nmol/L) and 5d (2-8nmol/L), more than 50% of pseudo-estrus cows can be excluded from the embryo transfer recipient list. (2) Birth rate from embryo transfer of cloned embryos through the progesterone monitoring is 7.1 fold higher than using the traditional monitoring method alone. Conclusion: The application of bovine serum progesterone monitoring can effectively improve the accuracy of cow reproductive cycle evaluation. It increases the effectiveness of suitable embryo transfer recipient cattle selection, as well as the accurate judgment of pregnancy status after SCNT embryo transfer in the recipient cattle. This practice can improve the utilization efficacy of recipient cattle selection, and the production efficiency of cloned cattle. It can be extended to other applications in the embryo engineering field as well as in the livestock production industry.
Objective: The voltage-gated calcium channel α2δ1 (isoform 5) subunit has been identified as a surface marker and therapeutic target for the tumor-initiating cells(TICs) of hepatocellular carcinoma(HCC). The anti-α2δ1 monoclonal antibody 1B50-1 could attenuate the growth of HCC in vivo by eradicating TICs. Hence, it is essential to construct the anti-α2δ1/CD3 bispecific antibody (BsAb) and evaluate its ability to kill liver cancer cells in vitro. Methods: The anti-α2δ1 scFv and anti-CD3 scFv were constructed by overlap PCR. Then the anti-α2δ1 scFv and anti-CD3 scFv were connected by (G4S1)3 linker and the bispecific antibody fragment was cloned into eukaryotic expression vector. After transfection of the plasmid into Expi 293F cells for 96 hours, the bispecific antibody was purified using nickel ion affinity chromatography. Flow cytometry was used to determine the binding properties of the BsAb for α2δ1 and CD3. Perkin Elmer Operetta High Content Imager was used to determine the ability of the BsAb directing cytotoxic T lymphocytes (CTLs) to kill Hep-12 liver cancer cell line which expresses high level of α2δ1. Enzyme-linked immunosorbent assay (ELISA) was used to detect the changes of hIL-2 and hIFN-γ secreted by CTLs during killing. Results: SDS-PAGE results show that the molecular weight of the anti-α2δ1/CD3 BsAb is consistent with theoretical value and the purified anti-α2δ1/CD3 BsAb is of great purity. Flow cytometry analysis reveals that the anti-α2δ1/CD3 BsAb binds specifically to the cells expressing α2δ1 or CD3. Cytotoxicity assay demonstrates that the BsAb can effectively mediate lysis of the α2δ+ Hep-12 cells (EC50=8pmol/L), while minimal cell lysis is observed for Hep-11 cells which express little α2δ1. Furthermore, the hIL-2 and hIFN-γ released by CTLs in the Hep-12 cell group during the killing process are higher than Hep-11 cell group (P<0.05). Conclusion: The anti-α2δ1/CD3 BsAb can effectively direct CTLs to kill the α2δ1+ Hep-12 cells in vitro, providing an alternative candidate of immunotherapy drug of liver cancer with bispecific antibodies.
Objective: To construct the phage library of anti-TNF-α (vascular endothelial growth factor) andto screen and express the nanobodies which have specificity and affinity with TNF-α. Methods: (1) The llama was immunized with TNF-α, and the total RNA of peripheral blood lymphocytes was extracted to construct a phage library, the clones having affinity with TNF-α were screened by multiple panning. (2) Then their molecular weight, pI and hydrophilicity were analyzed by ExPASy. And the VHH genes were cloned into the expression vector pNCS to construct the recombinant plasmids (pNCS-NbTNF-α) and to express these recombinant nanobodies (NbTNF-α) in E.coli DH5α. (3) The recombinant nanobodies were purified by Ni metal chelate affinity chromatography, followed by detection the specificity by enzyme linked immunosorbent assay (ELISA).Results: (1) Ten VHH gene fragments having affinity with TNF-α were obtained after phage library construction and panning. (2) Based on the bioinformatics analysis, it was found that eight nanobodies were hydrophilic proteins with molecular weights of 19.6-20.1kDa. All of NbTNF-α were expressed in E.coli DH5α in soluble form. (3) It was showed that five recombinant nanobodies, NbTNF-α-1, NbTNF-α-2, NbTNF-α-3, NbTNF-α-4 and NbTNF-α-5 could specifically bind to TNF-α. Conclusion: Eight nanobodies with specificity to TNF-α were screened and expressed in E.coli successfully, and five NbTNF-α showed good affinity with TNF-α, which could be possible candidates for anti-TNF-α drug.
Objective: To obtain hepatitis B core virus-like particles containing RGD targeting peptide and provide a new carrier for drug targeting nanometer delivery system. Methods: RGD-modified recombinant plasmid of hepatitis B virus was transformed into E. coli BL21 (DE3), and the optimal expression conditions of recombinant protein were investigated by single factor analysis and orthogonal test. Under the optimum expression conditions, the bacteria were cultured, collected after ultrasonic crushing, centrifuged, purified by Gel filtration chromatography, ion exchange and sucrose density gradient centrifugation, and the morphology and stability of RGD-HBc VLPs were identified by transmission electron microscopy.The purified RGD-HBc VLPs loaded the photosensitizer ICG into the inner part of the particle and injected it intravenously into 4T1 tumor-bearing mice of breast cancer to explore the targeting of recombinant RGD-HBc VLPs as a nano-delivery system. Results: RGD-HBc VLPs was efficiently expressed in the form of soluble protein at 32℃ and IPTG at 0.5mmol/L for 4h.After centrifugation with sucrose density gradient, the purity reached above 95%.The purified RGD-HBc VLPs were observed under transmission electron microscopy in uniform shape and size, with a diameter of about 32nm. Near-infrared fluorescence in vivo imaging confirmed the targeting of RGD-HBc VLPs as a nano-carrier. Conclusion: After expression and purification, RGD-HBc VLPs has a high expression level and uniform appearance, and near-infrared fluorescence in vivo imaging has a good targeting property, which not only provides a fast, accurate and convenient method for the visual diagnosis of tumor, but also provides a new carrier for future targeted immunotherapy.
The radiation environment exists everywhere in the living space, the results of animal experiments in recent years show that the harm of electromagnetic radiation are mainly concentrated on nervous system toxicity, inducing tumors (especially brain tumors, leukemia) and reproductive system damage. Radiation exposure has the characteristics of wide-area, concealed and cumulative effect. It acts on living organisms, causing a large amount of reactive oxygen species (ROS) in cells. By-products of normal aerobic physiological metabolism in the cells can also generate free radicals, thereby causing damage to the body. In other words, radiation can not only directly act on biological molecules and cause damage to the body, but also indirectly act on the body by acting on biological water and so on to produce free radicals. In order to detect the magnitude of radiation toxicity quickly and easily, there been established radiation biosensors. Engineered bacteria sensors carrying SoxR, RecA, Cda and SulA four promoters and enhanced green fluorescent protein (EGFP) fusion gene related to SOS reaction and oxidative stress reaction were constructed, that is, the promoter-reporter system. First, the four biosensors were treated with chemical damage agents, they all expressed a large amount of green fluorescent protein after stimulation, and then γ-ray irradiation was performed. According to the treatment, the sensor with the highest sensitivity was the RecA promoter engineering bacteria sensor under radiation. The promoter-reporter fusion gene obtained by PCR and overlap PCR, and inserted into the vector PUC19, then transformed into E. coli DH5α. After double-enzyme digestion and sequencing verification, the successful engineered bacteria sensors were disposed of chemical oxidant and physical radiation. The results showed that the four engineering bacteria sensors successfully responded to the oxidant hydrogen peroxide and physical radiation, and the green fluorescence intensity gradually increased with the increase of physical radiation dose (0-30Gy). Among them, the green fluorescence of RecA engineered bacteria sensor was the most obvious after stimulation compared with the other sensors. The use of synthetic biology methods to establish physical radiation sensors based on biological effects successfully, with simple preparation, visibility of results, meeting fast, wide range, online monitoring needs, solving the problem of excessive background value in chemical sensors. It has a good application prospect in the measurement of radiation, radiation on the ground and even in the space.
Objective: To investigate the effects of DNA vaccines on the fusion genes of SarA-Azami Green, IcaA-Azami Green, and IcaA-SarA-Azami Green (subsequently Azami Green was represented by AG) and their immune responses in mice.Method:Using the genome of S.aureus as a template, a series of reactions including SarA-AG, IcaA-AG, and IcaA-SarA-AG fusion DNA vaccines were constructed through a series of reactions such as PCR amplification, digestion, and ligation. After transfection into HeLa cells, the transient expression of DNA vaccine was observed under a fluorescent microscope. The cells after two weeks of successful transfection were subjected to genome extraction, and the integration of plasmids on chromosomes was detected by PCR. Recombinant plasmids were extracted using endotoxin-free plasmid extraction kits. BALB / c mice were immunized with AG, SarA-AG, IcaA-AG, and IcaA-SarA-AG. ELISA kits were used to detect mouse IgG antibodies, IL-2, IL-4, IL-13, IFN-γ and TNF-α secretion.Results: SarA-AG, IcaA-AG and IcaA-SarA-AG fusion gene DNA vaccines were successfully constructed. The transfection of the fusion gene DNA vaccine was observed under a fluorescence microscope, and the results showed green fluorescence. Genomic PCR results of the extracted cells showed that the target gene band did not appear. After immunizing mice, SarA-AG, IcaA-AG and IcaA-SarA-AG were able to induce mice to produce higher levels of IgG antibodies. Compared with the blank group and the empty vector AG group, the SarA-AG and IcaA-AG groups were able to secrete higher levels of IL-2 (P<0.001), IFN-γ (P<0.001), and TNF-α (P< 0.001), IL-4 (P<0. 01) and IL-13 (P<0. 01), while IcaA-SarA-AG group TNF-α (P<0.05), IL-4 (P<0. 05)) And IL-13 (P<0.05) were less secreted, but the difference was statistically significant. The secretion of cytokines increased significantly with the increase in the number of immunizations. There was no significant difference in cytokines between the blank group and the empty vector AG group.Conclusion: The DNA vaccines of SarA-AG, IcaA-AG and IcaA-SarA-AG fusion genes were successfully constructed and successfully expressed in eukaryotic cells. PCR verification results confirmed the safety of the DNA vaccine. DNA vaccine can induce humoral immune response and Th1 cell-based cellular immune response after immunizing mice, which has a good application prospect.
Surfactin, as a green biosurfactant, can be widely applied in various fields. However, the severe foaming arising in the fermentation process obstructed the industrial production of surfactin. Therefore, different strategies were explored to solve this problem in a 7L bioreactor. The results showed that excessive addition of antifoam would inhibit microbial growth and increase the production costs. The surfactin yield were 1.42g/L and 1.96g/L by using organic silicon and soybean oil as antifoams, respectively. However, foam fractionation in a modified bioreactor was more economical and effective for controlling the foaming and separating surfactin in situ. After fermentation coupled with foam fractionation, the surfactin yield was 2.39g/L. Based on the strategy of foam fractionation, the surfactin yield increased to 3.45g/L after controlling pH at 7. Moreover, it increased to 5.07g/L after controlling pH at 7 and DO≥20%. Furthermore, the surfactin yield increased to 6.04g/L by coupling foam fractionation with the regulation of pH, DO, and feeding (a constant rate of 1.39ml/min), which was 4.25 times higher than using antifoam. An efficient strategy is provided to control the foaming during fermentation and increase the surfactin yield, which can promote the industrial production of surfactin.
Diabetes is a chronic metabolic disease of hyperglycemia caused by various factors, which has developed into one of the epidemic diseases. Chemical anti-diabetic drugs can control the blood glucose and delay the progress of the disease, which needs to be taken for a long time to be effectively controlled. The current gold standard therapy for pancreas transplantation, but it has not been widely used because of the shortage of pancreas and the need for long-term use of immunosuppressive drugs. Mesenchymal stem cells (MSCs) are a kind of self-proliferation cells with the potential of multi-directional differentiation and paracrine characteristics. Recent studies have proved that MSCs have positive effects in the treatment of diabetes, and are considered as the ideal cell type for treatment of diabetes. Therefore, this review The molecular mechanisms and clinical research of MSCs for diabetes mellitus were briefly described.
Janus nanoparticles (JNPs) are used to describe a heterogeneous solid material composed of two different sides. Each side of JNPs is different in chemical nature and/or polarity, and can combine the characteristics and functions of different materials, which is difficult to achieve with homogeneous materials of the same kind. In recent years, major breakthroughs have been made in the preparation of JNPs, but the development direction of its application is still a challenging field, among which the research in the field of antitumor drug delivery systems is more prominent. The preparation method and application of Janus nanoparticles in drug delivery system, and puts forward the research prospects and possible challenges were mainly introduced.
Diarrheal Escherichia coli is a kind of foodborne pathogens that can cause human diarrhea disease. It will be effectively to narrow the scope of the outbreak disease if the source of diarrheal Escherichia coli could be confirmed at the first time. The establishment of a simple and efficient method for the detection and typing of diarrheal Escherichia coli is the key for food safety and epidemic control. The methods based on molecular biology technology were constantly standardized and normalized in order to satisfy the need of rapid detection and typing of diarrheal Escherichia coli.The recent advances in detection and typing of diarrheal Escherichia coli by molecular biological technique was reviewed. The principle, advantages and disadvantages of multiplex polymerase chain reaction, quantitative real-time PCR and isothermal amplification of nucleic acid were introduced in detail. It will provide reference for the selection of traceability methods for pathogenic bacteria, and have the great significance for the prevention and control of epidemic spread caused by pathogenic bacteria.
Caffeic acid and its ester derivatives like chlorogenic acid, rosmarinic acid, and caffeic acid phenethyl ester have important pharmacological activities such as natural antioxidant, antitumor, antiviral, and anti-inflammatory, and have broad prospects for medical development. Traditional extraction and chemical synthesis of caffeic acid and its derivatives from plants exist some problems such as low content, low extraction efficiency, high catalytic cost, and environmental pollution. In recent years, the study of heterologous synthesis of caffeic acid and its ester derivatives by microbes has been gradually carried out with the rapid development of synthetic biology.The recent advances in the biosynthetic pathway elucidations of caffeic acid and its ester derivatives and metabolic engineering strategies in heterologous microbes were summarized, and the current status as well as future perspectives were discussed.
Bibliometric analysis method is employed to analyze the current situation and development trend of global biotechnology research and its implications for the development of biotechnology research in China. The research results show that China has ranked first in the world in terms of the number of biotechnology article published in 2019, however, it still lags behind the developed biotech countries in Europe and the United States in terms of the quality of biotechnology articles, academic influence and international cooperation capacity. Then it analyzes the subject composition of global biotechnology research, and the cross-situation of each biotechnology research branch field. Finally, it analyzes the latest research keywords in each biotechnology subdivision field. Aiming at the current problems in the development of biotechnology research in China, related suggestions are put forward such as focusing on the quality of research results, promoting cross-disciplinary integration, strengthening international cooperation and exchanges, and conforming to future trends in scientific and technological development.
