Table of Content

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• Current Biotechnology
  Volume 10 Issue 4 25 July 2020
• Research Progress on Long Noncoding RNA in Regulating Fat Development and Metabolism
• YU Shulong¹, ZHANG Hao², LI Cencen^2*
• 2020, 10(4):  333-338.  DOI: 10.19586/j.2095-2341.2020.0038
• Abstract ( ) PDF (1100KB)( )
Long noncoding RNA (lncRNA) is a kind of RNA which is widely found in animals and plants, has a length of more than 200 nucleotides and cannot encode proteins. In recent years, with the continuous development of high-throughput genome sequencing technology, researchers have paid more and more attention to lncRNA. Through in-depth research on lncRNA, it has been proved that lncRNA plays an important role in many life activities such as cell differentiation, epigenetic, cell cycle regulation and so on, and the occurrence and development of many diseases are related to it. With the help of high-throughput sequencing or chip technology, it has been confirmed that many lncRNA are involved in the regulation of adipose tissue production, development and metabolism, and play an important role in the process of fat development. The study of lncRNA related to fat development can better understand the development and metabolic process of fat, and provide new methods for clinical treatment of metabolic diseases. Based on this, the researches on the  mode of action,  regulation of fat development of lncRNA and its influence on obesity-related metabolic diseases  were reviewed in order to provide theoretical guidance and direction for the research on fat development and metabolism.
• Advances on Anti-rabies Virus Neutralizing Antibodies
• BIAN Lun, LIN Guanfeng, WU Yingsong^*
• 2020, 10(4):  339-344.  DOI: 10.19586/j.2095-2341.2019.0099
• Abstract ( ) PDF (1097KB)( )
Rabies is a zoonotic infectious disease. The mortality rate of humans and animals is almost 100% after onset, and effective post-exposure preventive measures can reduce the risk of death to zero. According to the WHO recommended rabies post-exposure prophylaxis program, general rabies-exposed people need to be vaccinated, and in severe cases, they must be injected with anti-rabies virus neutralizing antibodies at the same time. Neutralizing antibodies that commonly used  are equine anti-rabies virus immunoglobulin and human anti-rabies virus immunoglobulin, but both have the risk of causing allergic reactions or blood diseases. Humanized anti-rabies virus neutralizing antibodies are expected to replace immunoglobulins for post-exposure prophylaxis because of their advantages of high safety, low cost, and mass production. The development of genetic engineering antibody technology has accelerated the process of antibody humanization. This article reviewed the development of anti-rabies virus neutralizing antibodies, the advantages and disadvantages of different types of neutralizing antibodies, and the future research expectations of neutralizing antibodies, which was expected to provide reference for research of the development of new generation rabies vaccine.
• Progress on Effect of  Transforming Growth Factor 5 in Developmental Dysplasia of the Hip
• YAO Jiawei, QIU Bo^*
• 2020, 10(4):  345-350.  DOI: 10.19586/j.2095-2341.2019.0134
• Abstract ( ) PDF (1102KB)( )
Developmental dysplasia of the hip (DDH) is an unstable joint disease mainly caused by structural deformities of acetabulum, proximal femur and joint capsule, which develops into dislocation of the hip. Dysplasia of cartilage in the hip joint, abnormal bones and tendons  can lead to structural deformity of the hip joint, and eventually lead to DDH. Therefore, early prevention and diagnosis is the key to DDH treatment. Researches show that DDH has genetic basis, and its susceptible genes include GDF5, HOXD9, COL2AL, PAPPA2, etc. Among them, growth differentiation factor 5 (GDF5) plays an important role in the proliferation and differentiation of chondrocytes, which is one of the hot spots in the treatment of DDH at present. Therefore, understanding the effect of GDF5 gene on cartilage development and differentiation is of great significance for the pathogenesis and treatment of DDH. Based on this, the progress of influence of GDF5 on DDH at the gene level was discussed recently at home and abroad, and the treatment schemes based on GDF5 such as intra-articular injection of recombinant human GDF5 were reviewed, in order to provide new strategies for clinical treatment of DDH.
• Progress in the Application of Vitamin Metabolism in Potato (Solanum tuberosum)
• SUN Hui, ZHANG Chunyi, JIANG Ling^*
• 2020, 10(4):  351-357.  DOI: 10.19586/j.2095-2341.2020.0048
• Abstract ( ) PDF (1126KB)( )
More and more people realize the importance of the relationship between the diet and the health, the contradiction between “quantities” and “quality” of food has become more prominent. Potato is the fourth most important  crop in the world, the construction of “Healthy China” and the  strategy of potato staple food have brought more challenges and opportunities to potato breeding and food processing. Potato is rich in the comprehensive nutritional value, especially vitamins, compared with other crops, but there is still a lack of potato varieties and products with good quality and quantity  in the market. This review focused on the latest research progress of plant vitamin metabolism, and the prospect and application value of the nutritional improvement in potato. These technical reserves for consumer oriented intensive nutrition will help understanding how to meet consumer preferences and the market demand.
• Application Progress of Atmospheric and Room Temperature Plasma Technology in Microbial Mutagenesis
• XU Huanhuan, ZHANG Hongbing^*, LI Huixuan, LI Lei
• 2020, 10(4):  358-362.  DOI: 10.19586/j.2095-2341.2020.0034
• Abstract ( ) PDF (1072KB)( )
Microorganisms are  important resources for human survival. In order to improve the production efficiency of microorganisms or endow them with new biological functions, mutagenesis by physical and chemical methods or site-directed mutagenesis by molecular biological techniques are required. Among the current methods of physical and chemical mutagenesis, atmospheric and room temperature plasma (ARTP) has become a favored new method due to its advantages of simple operation, mild conditions, high safety and rapid mutagenesis. Based on this, the principle of ARTP mutagenesis technology and its application in microbial mutation breeding were summarized, in order to provide reference for the mutation breeding related research of breeding superior microbial strains.
• Establishment of Event-specific PCR Detection Method of Transgenic Maize Line 2A-5
• WU Wenyan^§, LIU Xinxiang^§, ZHOU Miaoyi^*, LIU Jinxiang, LIU Ya
• 2020, 10(4):  363-370.  DOI: 10.19586/j.2095-2341.2020.0067
• Abstract ( ) PDF (6284KB)( )
According to the flanking sequence information of transgenic maize 2A-5, the optimal specific primer and TaqMan probe combination 2A-5-5-QF8, 2A-5-5-QR8, 2A-5-5-QP8 were designed and screened. The reaction system of the combinations was optimized as well, and the specific PCR detection method of transgenic event 2A-5 was established. The quantitative detection method of transgenic maize 2A-5 was studied by qRT-PCR and ddPCR. It was found that the combination of specific primers and TaqMan probe could complete the quantitative detection well. There was a high positive correlation between the content of transgenic components obtained by PCR reaction and the content of the sample. The specific PCR detection method and quantitative qRT-PCR and ddPCR detection method of transgenic maize 2A-5 in this study can detect 2A-5 with high specificity, accuracy and sensitivity, which is one of the effective methods to detect 2A-5 and its products accurately and efficiently in the future, and it also can provide good technical support for the safety supervision of genetically modified organisms in China.
• Identification of Pathogen Responsible for Anthracnose Disease of Postharvest Mango in Yunnan Province and its Biocontrol Bacterium Laboratory Screening
• YANG Yuan1§, GUO Yongfu1§, TANG Haozhi1, ZHANG Biao1, SHI Gonglin1, WANG Yating2, YANG Jun2, WEI Lanfang3*
• 2020, 10(4):  371-377.  DOI: 10.19586/j.2095-2341.2019.0090
• Abstract ( ) PDF (4003KB)( )
With the increasing demand for mango from Yunnan, the planting area of mango has been expanding, and new varieties are continuously introduced, thus causing the occurrence of anthracnose disease of mango in Yunnan Province to become more and more serious. In order to carry out targeted biological control of mango anthracnose, the pathogen of postharvest mango anthracnose was isolated by tissue block separation method. Morphological observation was used as preliminary identification, and the pathogen was verified according to Koch’s postulates. Subsequently, rDNA-ITS sequence and phylogenetic tree analysis were used to identify the taxonomic status of pathogenic bacteria. Finally, 5 kinds of biocontrol bacteria were used to conduct antagonistic tests on pathogenic bacteria. Through isolation and identification of the pathogen causing mango postharvest anthracnose, Colletotrichum gloeosporioides was determined to be the pathogen causing mango postharvest anthracnose in Yunnan Province. The sequence length of internal transcribed spacer (ITS) in this strain was 536 bp and the login number was MH744668. The five biocontrol bacteria had certain bacteriostatic to mango anthracnose pathogenic bacteria, and had good biocontrol development potential. Among them, the antibiotic L-44 had the best bacteriostatic effect, with the inhibition rate of 53.7%. The research results provided a new insight into biological control of mango postharvest diseases in Yunnan Province.
• Prokaryotic Expression of Ferritin and Recombinant Protein of Capsid Protein of IBDV and Extracellular Self-assembly of Nanoparticles
• DU Mengtan, ZHANG Weiye, LIU Xingjian, LI Yinyu, ZHANG Zhifang, HU Xiaoyuan^*
• 2020, 10(4):  378-385.  DOI: 10.19586/j.2095-2341.2020.0017
• Abstract ( ) PDF (5741KB)( )
Infectious bursal disease virus (IBDV) is the pathogen of infectious bursal disease (IBD). The high mortality of IBD causes severe economic losses to the global poultry industry. In recent years, the genetically engineered vaccines and DNA vaccines developed rapidly, bringing hope for more effective prevention and control of IBD. Ferritin can be assembled into stable multimers, which has been used as a candidate immunogen carrier for many diseases. In this study, the IBDV-VP2 and ferritin sequences were optimized according to the preference of Escherichia coli codons, and the fusion expression was carried out through the E. coli expression system, and the expression of the target protein was detected by mass spectrometry, Western blotting, SDS-PAGE, and transmission electron microscope. The fusion protein had been successfully expressed in the form of inclusion bodies in E. coli, and nano-sized particles of uniform size can be observed by transmission electron microscopy. This research can be used to explore the effect of ferritin self-assembly on improving the broad-spectrum and immunogenicity of IBDV vaccines, and lay the foundation for further research on its application mechanism as a drug and antigen carrier.
• Expression and Identification of Swine Fever Virus Envelope Protein E2 Fusion with Ferritin
• WEI Zhenzhen1, ZENG Zhen1, DU Mengtan2, LIU Xingjian2, ZHANG Zhifang2, HU Xiaoyuan2, LI Yinyu2, YI Yongzhu1*
• 2020, 10(4):  386-392.  DOI: 10.19586/j.2095-2341.2020.0028
• Abstract ( ) PDF (2825KB)( )
At present, the traditional inactivated and attenuated classical swine fever vaccines have some shortcomings, such as short immune protection period and strong virulence, and protein-derived subunit vaccines can solve these problems well.  24 subunits of ferritin can be self-assembled into stable multimeric nanoparticles, and become an ideal  antigen presentation carrier and vaccine development platform. Based on this, ferritin was combined with classical swine fever virus envelope protein E2 (protective antigen with high conservation) to construct a fusion gene E2-Fe, which was cloned into pET28a prokaryotic expression vector and then transformed into competent cells of Escherichia coli BL21 (DE3) to explore the induction conditions and express it efficiently. Then the expressed fusion protein was purified by nickel column, analyzed by mass spectrometry, verified by Western-blotting, and detected by electron microscope. The results showed that the fusion gene E2-Fe had been successfully cloned into pET-28a vector, and the best expression condition of fusion protein E2-Fe was induced by 0.50% lactose for 6 h. Mass spectrometry analysis and Western-blotting showed that the size of the fusion protein was about 51 kD, which was consistent with the theoretical value. The diameter of nanoparticles observed by electron microscope was about 20 nm. The results showed that the envelope protein E2 of classical swine fever virus fused with ferritin was highly expressed in E. coli, and a considerable amount of self-assembled nanoparticle antigens could be obtained after purification by nickel column, which broadened the research ideas for studying new classical swine fever vaccines.
• Study on Effect of Hydrogen on Excitatory Conduction in Rats
• ZHANG Xiaokang, YI Yang, XIE Fei, ZHANG Zhao, MA Shengnan, ZHAO Pengxiang, MA Xuemei^*
• 2020, 10(4):  393-399.  DOI: 10.19586/j.2095-2341.2020.0018
• Abstract ( ) PDF (1820KB)( )
Hydrogen is a gas molecule with important biological functions, which can be used for the treatment and improvement of neurodegenerative diseases, depression, sleep disorders and drug addiction withdrawal symptoms. It is generally believed that it is related to the selective oxidation resistance of hydrogen, but the regulatory mechanism of hydrogen on neural function has not been explained clearly. In order to explore the regulation mechanism of hydrogen on neural function, the action potential changes of cortical neurons in cerebral slices of rats exposed to hydrogen transiently and cortical neurons in cerebral slices of rats that drinking hydrogen-rich water for 8 weeks were detected by patch clamp technique to determine whether hydrogen intervention can affect the conduction of nerve excitation. Liquid chromatography-mass spectrometry (LCMS) was used to detect the changes of neurotransmitters in cerebral slices of rats drinking hydrogen-rich water (eight weeks) in order to further explore the specific mechanism of hydrogen affecting nerve excitation conduction. The results showed that compared with the control group, the threshold voltage, action potential interval and input impedance of rat cortical neurons in the hydrogen treatment group had significant difference (P<0.05). The resting membrane potential  in the hydrogen treatment group increased, and the threshold of nerve cell burst action potential increased, which indicated that hydrogen might affect the opening and closing of ion channels in nerve cell membrane, and hydrogen treatment could significantly reduce the excitability of cortical neurons. After drinking hydrogen-rich water continuously for eight weeks, the cerebral cortex of rats also showed a decrease in excitability, and LCMS showed no significant change in neurotransmitter content. Research suggested that hydrogen may regulate the excitability of nerve cells by changing the change of charge difference inside and outside cells or directly affecting the opening or closing of sodium and potassium plasma channels on the surface of nerve cells.
• Effects of Molecular Hydrogen on Liver Cancer Cells Huh7
• SHANG Lei, LI Jiala, SU Zehua, XIE Fei, MA Xuemei^*
• 2020, 10(4):  400-408.  DOI: 10.19586/j.2095-2341.2020.0024
• Abstract ( ) PDF (6171KB)( )
The effects of molecular hydrogen on various diseases such as cancer are predictable. It is simple to obtain, easy to use and free of side effects. In order to clarify the effect of molecular hydrogen on hepatoma cell Huh7, the effect of molecular hydrogen on Huh7 cells was studied from the aspects of cell viability, cell cycle and apoptosis, cell neoplasm formation, cell migration and invasion. The results of cell viability test (CCK-8 method) showed that molecular hydrogen had obvious inhibitory effect on Huh7 cell activity. In the experiment of cell neoplasm formation, the clone ball experiment showed that the number of cell globules in the hydrogen-containing medium group (H) decreased, while the diameter of clone ball decreased significantly (P<0.000 1). The plate cloning experiment showed that the number of cell clones in group H also decreased significantly (P<0001), indicating that molecular hydrogen can inhibit the dryness of Huh7 cells. Molecular hydrogen also had obvious effects on Huh7 cell cycle, and had the effects of promoting apoptosis and inhibiting cell migration and infiltration. At the same time, hydrogen molecules could also inhibit the expression of vimentin in cells. Studies showed that hydrogen molecules reduced the activity and proliferation ability of Huh7 cells, weakened the dryness of Huh7 cells, inhibited the cell invasion and migration ability, and reduced the expression of cellular vimentin, which provided a certain experimental basis for the application of hydrogen molecules in the prevention and treatment of liver cancer.
• The Effect of Hemin-induced Erythroid Differentiation in K562 Cells on Cell Cycle
• MA Yige1, WANG Bingrui1, GAO Jie1, LIU Jinhua1, TONG Jingyuan1, LIU Lijun2*, SHI Lihong1*
• 2020, 10(4):  409-416.  DOI: 10.19586/j.2095-2341.2020.0033
• Abstract ( ) PDF (7343KB)( )
Cell proliferation kinetics based on the measurement of cell cycle length. In this paper, 30 μmol·L-1 Hemin induced erythroid differentiation of human chronic myeloid leukemia K562 cells. Hemin-induced K562 erythroid differentiated cells were detected the effect on the percentage of cell cycle phases by using 5-bromodeoxyuridine (BrdU) and 7-AAD double staining. The percentage of each phase of K562 cells were used as  control. It was found that Hemin-induced K562 erythroid differentiated cells had no significant effect on the relative value of cell cycle. Interval staining combined with flow cytometry were used to analyze the dynamic change of BrdU positive cell population so that the doubling time and cell cycle duration of erythroid differentiated cells of K562 cells were calculated. According to the measurement results, the total doubling time of K562 cells was about 20 hours, which was consistent with the calculation of the doubling time by growth curve methods. The cell cycle doubling time of K562 cells induced by Hemin was about 23 hours. The doubling time of K562 cells induced by Hemin was not significantly different from K562 cells. Therefore, Hemin-induced erythroid differentiation of K562 cells had no significant effect on the absolute value and relative value of cell cycle.
• Expression and Prognostic Significance of SCHIP1 in Acute Myeloid Leukemia: Analysis Based on  Bioinformatics
• XU Jie, WANG Kefei, WEI Xiaojing, GONG Lixin, JIAO Yang, QIU Lugui, HAO Mu^*
• 2020, 10(4):  417-425.  DOI: 10.19586/j.2095-2341.2020.0019
• Abstract ( ) PDF (9206KB)( )
The study was aimed to  investigate the expression and clinical significance of schwannomin interacting protein 1 (SCHIP1) in patients with acute myeloid leukemia through data mining and bioinformatics analysis. First of all, a meta-analysis was performed on all AML data sets including  the Oncomine database,  which aimed to screen the target gene SCHIP1 and further analyze its expression changes in AML patients. Then, AML dataset source files containing survival information were downloaded from GEO database to analyze the prognostic effect of SCHIP1 on disease. And the expression of SCHIP1 among subgroup and the correlation with FLT3 gene mutation, PML/RARα fusion and RAS activation were analyzed using TCGA database. Ultimately, GEPIA2 was used to verify the expression and prognostic significance of SCHIP1 and its correlation with FLT3 and PML gene expression. Results showed that 44 AML datasets were included in Oncomine database, with a total of 3 534 sample data. And a total of 1 188 samples from 5 datasets contained mRNA expression data of "Cancer vs. Normal", and a meta-analysis of these samples showed that SCHIP1 was the 17th most highly expressed molecule. Then, survival analysis showed a negative correlation between SCHIP1 expression and overall survival in AML patients. And subgroup analysis showed that the expression of SCHIP1 in M0/M1/M2 was higher than that in M3/M6, regardless of age, gender or race. In addition, correlation analysis showed a weak correlation between SCHIP1 and FLT3 gene mutation, but no significant correlation with PML/RARα fusion, RAS activation and other risk factors. These results showed that SCHIP1 is highly expressed in acute myeloid leukemia (AML), and its high expression is significantly negatively correlated with overall survival of patients.Therefore, it can be used as a prognostic biomarker for the disease, and it is expected to become a precise therapeutic target for AML.
• Study on Optimum Process Conditions Immobilized Cellulase with Carbon Nanotubes
• LI Lijuan¹, XIA Wenjing², MA Guiping¹
• 2020, 10(4):  426-431.  DOI: 10.19586/j.2095-2341.2020.0021
• Abstract ( ) PDF (2327KB)( )
Cellulase has been widely used in environmental protection, medicine, food and other fields. However, the widespread use of cellulase was limited by its relatively high production costs and low biological activity. In this study, in order to provide a method for immobilizing cellulase that allowed the enzyme to be reused in multiple cycles, multi-walled carbon nanotubes was used as carriers to immobilize cellulase for the first time. The conditions for immobilization of enzyme with multi-walled carbon nanotubes were studied. An orthogonal experimental design was applied to optimize critical conditions for the immobilization process. The structures of MWCNTs, cellulase and immobilized cellulase were characterized by Fourier transform infrared (FT-IR) spectroscopy. The results showed that the optimum process conditions for immobilized enzyme were as follows: enzyme concentration 5 mg·mL-1, temperature 40 ℃, pH 5.0, immobilization time 3 h, and the immobilization of cellulase was confirmed successfully onto the multi-walled carbon nanotubes by Fourier transform infrared spectroscopy.
• Study on Protoplast Fusion and Breeding of Clostridium acetobutylicum
• WANG Huan, WU Fang, NIU Kun^*
• 2020, 10(4):  432-437.  DOI: 10.19586/j.2095-2341.2020.0029
• Abstract ( ) PDF (1943KB)( )
The study was airmed to improve the butanol tolerance of Clostridium acetobutylicum and increase the conversion rate of total sugar in culture medium to produce butanol by the protoplasmic fusion method. The effect of  concentration of lysozyme and its action time, the type of regenerative medium, the time of thermal death at 55 ℃, different PEG molecular weights and the action time, Ca2+, Mg2+ additions with different amounts on Clostridium acetobutanol protoplasm preparation, fusion, regeneration was studied. We obtained a set of comparative system of Clostridium acetobutanol protoplasm fusion conditions. And we detected the solvent production capacity of the fusion bacteria by gas chromatography and calculated the total sugar conversion rate. Results showed that, the total sugar conversion rate of the final obtained strain 215I was 34.7% higher than the original strain, and the butanol production capacity was 32.2% higher than the original strain, and one fusion strain was found to produce new substances. The protoplasmic fusion method is promising in the breeding of Clostridium acetobutylicum, and the strains obtained by fusion provide a basis for further experiments.