Current Biotechnology

Progress in Research and Development of Antibodies for Treatment of Respiratory Infectious Diseases
YANG Gaosong¹, MA Dongjie^2*
2020, 10(5): 441-447. DOI: 10.19586/j.2095-2341.2020.0026
Abstract ( ) PDF (1180KB)( )

Respiratory diseases affect millions of people all over the world. The main lesions occur in trachea, bronchus, lung and chest. The mild ones have cough and chest pain, while the severe ones have dyspnea, hypoxia and even respiratory failure, resulting in a variety of complications leading to severe disability and even death. Clinical use of therapeutic antibodies has opened up a new way for the treatment of lung cancer, asthma and various respiratory infectious diseases. At present, dozens of antibodies (Abs) have been approved into the market, and more antibodies are under clinical development. Most of these Abs are aimed at asthma, lung cancer, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and respiratory infectious diseases. Among them, the outbreak of respiratory infectious diseases has the characteristics of rapid spread and strong infectivity, which often causes global concern, such as coronavirus disease 2019 (COVID-19), which is now raging all over the world. A variety of Abs for respiratory infectious diseases provides new strategies for their clinical treatment. Based on this situation, Abs, which have been approved and are being developed clinically, were reviewed. The molecular mechanisms, advantages and development trend of antibody therapy were summarized in order to provide references for the research and development of Abs in the treatment of respiratory infectious diseases.

Research Progress of Glycosylation Engineering of Protein Drug
DU Li, LIU Xiaozhi, WEI Jinshuang, GAO Jian^*
2020, 10(5): 448-455. DOI: 10.19586/j.2095-2341.2020.0027
Abstract ( ) PDF (1170KB)( )

Mammalian and non-mammalian protein expression systems have been used in the production of recombinant glycoprotein drugs. Glycosylation is very important for the research and development of biopharmaceuticals, which has an important impact on its efficacy, half-life and antigenicity. The aim of glycosylation engineering is to produce N- and O-linked glycosylated protein drugs with clear components and uniform structure. Recent studies on N-glycosylation have shown that homogeneous recombinant N-glycans can be expressed in mammalian and non-mammalian expression systems. Compared with N-glycosylation, O-glycosylation is still in its infancy. The first glycosylation engineering monoclonal antibody has been approved for marketing in the United States and Japan. This paper reviewed the research progress of glycosylation engineering of recombinant protein expression system, including the progress on N-glycosylation and O-glycosylation, which was expected to provide reference for glycosylation engineering of protein drug research.

Charge Heterogeneity and Process Control of Recombinant Monoclonal Antibodies
WANG Huan^§, NIU Kun^§, JIANG Yifan, DONG Jing^*
2020, 10(5): 456-462. DOI: 10.19586/j.2095-2341.2020.0050
Abstract ( ) PDF (1999KB)( )

Recombinant monoclonal antibody drugs are mostly post-translationally modified and the types are complex and diverse. Therefore, quality control during the development process is particularly important. Among them, charge heterogeneity is a critical quality attribute, which may affect the efficacy of biological products, and may even bring unexpected side effects, which will affect the safety and effectiveness of the drug. Therefore, it is necessary to focus on and pay attention to the development of mAb regulation. Post-translational modification of monoclonal antibody drugs is the main cause of charge heterogeneity. Therefore, control of charge heterogeneity is an important challenge for the development of biopharmaceutical technology. This article reviewed the characterization methods of charge heterogeneity, and summarized the post-translational modifications of proteins that can cause charge heterogeneity according to its classification. At the same time, the paper described the safety and effectiveness of different charge heterogeneity for antibody drugs. Finally, the latest progress of charge heterogeneity process control strategy in process development was summarized. We hope to give inspiration to researchers in the development and quality of biopharmaceutical processes.

Mechanism Progress on Fibroblast Growth Factor 21 Analogue Treating Atherosclerosis
OUYANG Man
2020, 10(5): 463-469. DOI: 10.19586/j.2095-2341.2020.0037
Abstract ( ) PDF (2155KB)( )

Atherosclerosis is the main cause of coronary heart disease, cerebral infarction and peripheral vascular disease. Incidence is increasing, which threatens human life and health seriously. Lipid metabolism disorder is the pathological basis of atherosclerosis. Fibroblast growth factor 21 (FGF21) is an endocrine factor of the FGF family. It increases glucose uptake, modulates lipid metabolism, and sensitizes insulin response in metabolically active organs, including the liver and adipose tissue. Emerging evidence showed a strong correlation between circulating FGF21 levels and the incidence and severity of atherosclerosis. However, the short plasma half-life of native FGF21 and its propensity to aggregate in vitro have limited its clinical application. Recently, breakthrough progress has been made in FGF21 analogues. The present review summarized the physiological roles of FGF21, and discussed major breakthroughs and limitations of FGF21 mimetic-based therapeutic strategies for treating atherosclerosis, which was expected to provide theoretical basis for the development of new FGF21 protein drugs.

Progress on Newcastle Disease Vaccine
YAO Shunyu, ZHANG Lilin*
2020, 10(5): 470-478. DOI: 10.19586/j.2095-2341.2020.0052
Abstract ( ) PDF (1198KB)( )

Newcastle disease (ND) is one of the viral diseases of poultry, which can cause damage to nervous system, digestive system and respiratory system of poultry, with a mortality rate as high as 30%, which severely restricts poultry breeding. Therefore, it is of great economic significance to study ND. Rational use of vaccines is the main method to prevent and control Newcastle disease. Since 1950, live attenuated and inactivated Newcastle disease vaccines have been widely used, and then vector vaccines have entered the commercial application stage. In addition, the choice of vaccine adjuvant and the optimization of delivery route have also entered the researchers’ field of vision. Based on this situation, the research and development status and prospect of traditional vaccine, vector vaccine and virus-like particle vaccine for Newcastle disease were analyzed, and the application progress of nanoparticles and new immune adjuvant in the research and development of Newcastle disease vaccine was introduced. Then the common commercial vaccines of Newcastle disease at home and abroad were summarized, aiming to provide references for developing more efficient and cheap Newcastle disease vaccine, and further control the spread of Newcastle disease epidemic.

Progress of Vitamin E Biofortification in Plants
YAO Xinglan, WANG Lei^*, ZHANG Lan^*
2020, 10(5): 479-486. DOI: 10.19586/j.2095-2341.2020.0046
Abstract ( ) PDF (2264KB)( )

Vitamin E is a kind of fat-soluble antioxidant and it is synthesized only by photosynthetic organisms. Vitamin E is an important and indispensable vitamin for human and livestock. Due to the low content of vitamin E in plants, most of people are in a state of chronic deficiency of vitamin E——“hidden hunger”. Synthetic vitamin E is supplemented in the feed for optimal animal growth. Therefore, increasing the content of vitamin E in plants is one of the important ways to improve vitamin E deficiency. Starting from the biosynthetic pathway of vitamin E, this paper summarized the influence of key enzymes expression and precursors content on vitamin E in detail, pointed out that the biofortification effect of tocotrienol and α-tocopherol was efficient, while the increase of tocopherol content was limited. Furthermore, the reasons for the limitation of vitamin E biosynthesis and other metabolic pathways that may affect vitamin E synthesis were discussed from the genetic point of view. Finally, a new idea was proposed for vitamin E biofortification in the future by combining with the regulatory factors that may affect the biosynthesis of vitamin E and the transport of its precursors.

The Role of Selenium and Folic Acid in Antiviral Therapy and Their Application Prospects in Plants
SUN Hui, ZHANG Chunyi, JIANG Ling^*
2020, 10(5): 487-494. DOI: 10.19586/j.2095-2341.2020.0074
Abstract ( ) PDF (1488KB)( )

During the treatment of the coronavirus disease 2019 in 2020, scientists have found out that better effect of antiviral treatment was associated with selenium and folic acid levels in human. The results indicated that the diet with high levels of selenium and folate can effectively enhance human immune function and to cope with the impact of viral diseases. One of the main tasks of plant biofortification is to ensure the intake of selenium and folate effectively through diet. However, there were few reports on the combined biofortification of selenium and folate. This paper reviewed the ways and mechanisms of selenium and folic acid in the treatment of antiviral, as well as the application prospects of selenium and folate in plants, so as to provide the theoretical support for improving nutritional quality and creating crops, vegetables and fruits rich in selenium and folate.

Research and Application Progress of Exocellulase
ZHANG Qingcui, SHI Yali^*, LIU Anli, HU Jianhua, LI Yongli, SUN Yachao, HE Kexin, XIA Ting, BAO Yanbin
2020, 10(5): 495-502. DOI: 10.19586/j.2095-2341.2020.0064
Abstract ( ) PDF (1154KB)( )

Cellulases mainly include three categories: exocellulase, endocellulase and β-glucosidase. Among them, exocellulase is widely used in various industrial production because of its high activity, good tolerance and wide sources. Based on this, the classification, sources and biochemical characteristics of exocellulase were mainly expounded, and the new technologies related to the screening of exocellulase and the analysis of its hydrolysate were introduced. The application progress of exocellulases in paper industry, food processing industry, pharmaceutical industry, textile industry, energy regeneration and other industrial fields was summarized, in order to promote the further study and industrial application of exocellulases.

The Transcriptome Analysis of Wheat Dwarf Mutant jm22d Responding to GA Treatment
FANG Hanshun1,2, XIE Yongdun2, ZENG Weiwei2, GUO Huijun2, XIONG Hongchun2, ZHAO Linshu2, GU Jiayu2, XU Yanhao1*, LIU Luxiang2*
2020, 10(5): 503-516. DOI: 10.19586/j.2095-2341.2020.0108
Abstract ( ) PDF (13053KB)( )

In order to enrich the genetic diversity of dwarf gene resource in wheat, we irradiated Jimai 22 by γ-rays and obtained a GA insensitive dwarf mutant, named as jm22d. Investigation of plant height and stem cell showed that jm22d was about 53±1.8 cm, 20 cm shorter than that of wild type (WT). The internodes number of jm22d was 4, one less than WT plants, and the internode length of the mutant was significantly shorter than that of WT. The endogenous gibberellin content in jm22d leaves was higher than WT, while endogenous gibberellin content in stem was significantly lower than WT, suggesting that the dwarfing of jm22d might be caused by the abnormal gibberellin transportation pathway. Both mutant and WT seedlings were treated with gibberellin to study the mechanism that jm22d response to gibberellin, and samples were collected from 0 (D0),1 (D1) and 3 days (D3) respectively. Transcriptome analysis was conducted to analyze the dwarfing mechanism of jm22d. Comparing to WT, totally 696 up-regulated genes and 1 067 down-regulated genes were identified in jm22d. Among of them, 62 genes were up-regulated and 349 genes were down-regulated at all of the three time points. The determination of chlorophyll content showed that the chlorophyll content in jm22d decreased with the extension of gibberellin treatment and the differentially expressed genes (DEGs) was significant enriched in photosynthesis-antenna proteins, porphyrin and chlorophyll metabolism, linoleic acid metabolism and zeatin biosynthesis pathways. Gibberellin can inhibit the accumulation of chlorophyll content in jm22d. Five differentially expressed genes were identified in plant hormone signal transduction pathway involved in auxin and cytokinin metabolic pathways (TraesCS2B01G582300, TraesCS2B01G600800, TraesCS2B01G556600, TraesCS2B01G630000 and TraesCS6B01G4396009) through KEGG analysis, and they were significant up-regulated in jm22d. This may be the main reason for the dwarfing of jm22d. The results of this research provide an important reference for the mechanism of wheat dwarf mutants.

Identification of Two Sec-dependent Cellulases in Erwinia amylovora and Gene Expression Analysis During Infection of Immature Pear Fruit
YAN Hailin1,2,3, ZHU Zongcai1,2,3, ZHANG Wangbin1,2, DU Peixiu3, ZHANG Chao3, ZHAO Wenjun4, LI Weimin3*
2020, 10(5): 517-523. DOI: 10.19586/j.2095-2341.2020.0084
Abstract ( ) PDF (2660KB)( )

In this study, by using bioinformatics analysis and Escherichia coli-based alkaline phosphatase (PhoA) gene fusion system, we identified two Sec-dependent secreted cellulases, an endoglucanase EAMY_3602 and a β-glucosidase EAMY_1236, from the whole genome of Erwinia amylovora. The cell suspension of E. amylovora strain DSM17948 was inoculated into the young pear fruits, quantitative detection of gene expression at 0, 8 and 24 hpi (hours post-inoculation), respectively, which showed that the mRNA expression levels of Ea3602, the coding gene of EAMY_3602, were continuously decreased at 8 and 24 hpi, with no significant difference when compared with that at 0 hpi. The transcript levels of Ea1236, the coding gene of EAMY_1236, at 8 and 24 hpi were 2.43 and 3.26 fold higher than that at 0 hpi. Research results showed that the Sec-dependent secreted endoglucanase EAMY_3602 had no implication in plant infection, but β-glucosidase EAMY_1236 probably acted as a virulence factor that plays role in the process of E. amylovora infecting the immature pears.

Using the Runx1c-mNeongreen Reporter Cell Line Derived from the Human Embryonic Stem Cells to Track the Definitive Hematopoiesis
ZHU Yaoyao, ZHANG Shuo, WANG Zhengyu^*
2020, 10(5): 524-533. DOI: 10.19586/j.2095-2341.2020.0080
Abstract ( ) PDF (9956KB)( )

Runt-related transcription factor 1c (Runx1c) is closely related to definitive hematopoiesis and the production of hematopoietic stem cell (HSC). In order to track the dynamical expression of Runx1c during early hematopoietic development, Runx1c-mNeonGreen reporter cell line derived from human embryonic stem cell (hESC) was efficiently constructed by CRISPR-Cas9 technology combined double-cut plasmid vector with the transient transfer of B-cell lymphoma-extra large (BCL-XL) gene. It was identified and verified by PCR and Sanger sequencing, and the results showed that the construction was successful. Subsequently, immunofluorescence technique, real-time fluorescence quantitative PCR technique, flow cytometry, teratoma assay and digital karyotype analysis technique were used to verify that the reporter cell line still had hESC-like pluripotency and no chromosome abnormality or variation occurred. In addition, the expression of Runx1c in human early hematopoietic development was tracked by flow cytometry through hESC in vitro monolayer induced hematopoietic differentiation. The results showed that the reporter cell line could dynamically track the expression of Runx1c in human early hematopoietic development. The research results provided a new idea for elucidating definitive hematopoiesis and the possibility of producing functional blood cells in vitro.

Early Hyperbaric Oxygen Intervention Improved Survival Rate Under Cold Stress and Accelerated Wound Healing Process in Murine Model
WANG Limin1, LIU Shibo1, GUO Dazhi2, PAN Shuyi2, WANG Zhen1, GUO Boyuan1, WANG Hui1, ZHANG Xujuan1, ZHAO Pengxiang1*, XIE Fei1, MA Xuemei1
2020, 10(5): 534-540. DOI: 10.19586/j.2095-2341.2020.0051
Abstract ( ) PDF (6571KB)( )

Open wounds subjected to cold stress are an extremely dangerous form of battlefield injury, and are threating people suffering from exposure in wild areas. Among emergency treatments, hyperbaric oxygen (HBO) therapy has been found to be safe and effective in wound healing. However, the best intervention time for HBO is still unclear. In this study, by using a murine dorsal full-thickness wound model under cold stress, we compared three kinds of intervention strategies for HBO: 0, 24 and 48 hours after wounding. The results showed that immediate (0 h) HBO treatment had the best outcomes to reduce death rate (0-hHBO 33% vs. control 100%) on day 5, when wound closure rates were 85%. Further whole blood testing and immunochemical detection revealed 0-hHBO therapy improved hematological indicators and exerted anti-apoptotic effects, especially in epidermal stem cells. The results provide important clues for clinical application of HBO.

Inhibitory Effect of Hydrogen Inhalation on Acute Inflammation in Rats with Traumatic Brain Injury
JU Fangdi1§, XIE Fei1§*, GUO Dazhi2§, ZHAO Qinghui1, HE Jin1, YAO Tingting1, ZHAO Pengxiang1, PAN Shuyi2*, MA Xuemei1*
2020, 10(5): 541-549. DOI: 10.19586/j.2095-2341.2020.0056
Abstract ( ) PDF (3983KB)( )

In order to investigate the effect of hydrogen inhalation on inflammatory response in acute phase of traumatic brain injury (TBI) in rats, 6-week-old male SD rats were randomly divided into sham operation group, TBI group and hydrogen inhalation treatment group. The level of serum cytokines was detected by suspension chip technology at 2, 6 and 24 h after TBI, and the neuroprotective effect of hydrogen inhalation was evaluated by modified neurological severity score (mNss) at 24 h after TBI. Meanwhile, the brain tissue was taken for Nissl staining analysis and serum biochemical indexes were detected. The results of mNss score showed that the neurological function of TBI rats was significantly improved within 24 h after hydrogen inhalation. The protective effect of hydrogen inhalation on neurons was further confirmed by Nissl staining. The results of serum cytokines detection showed that hydrogen inhalation could exert inhibitory effect on acute inflammation induced by TBI, evidenced by remarkable reduction in seven pro-inflammatory cytokines in serum at 2 h after TBI. In addition, hydrogen inhalation markedly decreased serum levels of cardiac and hepatic biomarkers, suggesting that hydrogen inhalation could attenuate cardiac and hepatic function impairment in the acute phase of TBI. Studies suggested that hydrogen inhalation may play a neuroprotective role by inhibiting the inflammatory response in the acute phase of TBI.

Effect of Bee Pupa Polypeptide on the Immune Activity of Macrophage RAW264.7
ZHAO Jie1, ZHANG Weijie2, CHEN Yao3, XIANG Qingfang1, ZHAO Ting1, MAO Guanghua3, FENG Weiwei3, YANG Liuqing1*
2020, 10(5): 550-556. DOI: 10.19586/j.2095-2341.2020.0055
Abstract ( ) PDF (2499KB)( )

Bee pupa polypeptide has attracted wide attention because of its rich nutritional value and biological activities such as immune enhancement, anti-tumor and anti-oxidation. However, there is no report on the immunomodulatory activity of purified components of bee pupa polypeptide in vitro. In order to explore the effect of bee pupa polypeptide on the immune activity of macrophage RAW264.7, BPP-21, a purified component of bee pupa polypeptide, was taken as the research object, and the effects of BPP-21 at different concentrations (12.5, 25, 50, 100 and 200 μg·mL-1) on cell viability, phagocytosis, cytokine secretion, NO secretion and oxidative stress indexes of RAW264.7 were studied. The results showed that BPP-21 had no significant inhibitory activity on RAW264.7 cells within the concentrations ranged from 12.5 to 200 μg·mL-1, and significantly increased nitric oxide (NO) and interferon-γ (IFN-γ) secretion (P<0.05). In the range of 25~200 μg·mL-1, BPP-21 could significantly increase phagocytic activity, as well as interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) secretion (P<005). In the range of 50~200 μg·mL-1, the activity of superoxide dismutase (SOD) in cells was significantly increased (P<005). The results showed that BPP-21, a purified component of bee pupa polypeptide, could enhance the immune activity of RAW264.7 macrophages, which provided a theoretical basis for the research and development of bee pupa polypeptide immunomodulator.

Qualitative and Quantitative Analysis of Tilapia Tissues Based on Low-field Nuclear Magnetic Resonance T1-T2 Spectroscopy
LIU Hongliang1, DING Qianwen1, TEAME Tsegay1, HAO Qiang1, WANG Anran1, MA Deming2, RAN Chao1, YANG Yalin1, ZHANG Zhen1*, ZHOU Zhigang1*
2020, 10(5): 557-563. DOI: 10.19586/j.2095-2341.2020.0049
Abstract ( ) PDF (2881KB)( )

The detection of morphology index is an important part of routine nutrition evaluation of fish. At present, the method of analyzing fish tissue-weight ratio will cause damage to fish, and the sampling process is complex, so it is urgent to analyze fish tissue-weight ratio by rapid non-destructive detection technology. Low-field nuclear magnetic resonance (NMR) technology has the characteristics of rapid and nondestructive detection. Low-field NMR T1 spectroscopy has been used to detect the volume of adipose tissue in mice, but low-field NMR T1-T2 spectroscopy has not been used for qualitative and quantitative analysis of fish tissues and organs yet. Based on this, the muscle tissue, abdominal adipose tissue, liver tissue and intestinal tissue of tilapia after separation were scanned and analyzed by using low-field NMR T1-T2 spectroscopy independently and mixed. It was found that the muscle tissue and abdominal adipose tissue of tilapia could be separated by using low-field NMR T1-T2 spectroscopy, but liver tissue and intestinal tissue could not be distinguished. Furthermore, a quantitative analysis model was established for muscle tissue and adipose tissue which could be separated, and the correlation between tissue signal strength and tissue weight of tilapia was analyzed. The results showed that the correlation (R2) of muscle tissue was 0.974 3, and that of abdominal adipose tissue was 0965 0. The reliability of the quantitative analysis model of muscle tissue was verified by tilapia in vivo, and the muscle signal size in vivo was converted into muscle tissue weight and its correlation with the whole fish weight was analyzed. The results showed that the correlation (R2) between muscle tissue weight and body weight was 0.806 9. The research showed that the low-field NMR T1-T2 spectroscopy could be used to quantitatively analyze the content of muscle tissue in fish quickly and non-destructively.

Isolation, Identification of an Acetic Acid Producing Strain from Tomato Surface and the Optimization of Acid Producing Conditions
ZHONG Caixia¹, TIAN Jiaxue², WANG Xiaoqi², WEN Tong^2*
2020, 10(5): 564-569. DOI: 10.19586/j.2095-2341.2020.0058
Abstract ( ) PDF (2761KB)( )

Acetic acid bacteria plays a key role during vinegar fermentation. The strains with excellent performance are of great significance for improving the vinegar quality. In this study, acetogenic bacteria isolated from tomato surface were identified by physiological and biochemical index identification and 16S rRNA coding gene blasting. Then the culture conditions of strain which identified as Acetobacteraceae were optimized by orthogonal experiments and single factor changes. The results showed that among the three strains of acetic acid producing bacteria, BQ-1 was identified as Acetobacteraceae. In the medium with yeast extract as the main nitrogen source and sucrose as the main carbon source, the highest acid yield of BQ-1 was 18.23 g·L-1. Because of its strong growth ability on tomato surface, the strain is expected to be used in tomato vinegar producing.

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