Preparation of human monoclonal antibody against SARS-CoV-2 spike protein using single B cell / 中国生物制品学杂志

@#Abstract：Objective To prepare human monoclonal antibody against spike protein（S protein）of severe acute respiratory syndrome coronavirus 2（SARS⁃CoV⁃2）by using single B cell，and determine its neutralizing activity. Methods Venous blood with high antibody level was collected from people immunized with inactivated SARS⁃CoV⁃2 vaccine（Vero cells） twice，of which peripheral blood mononuclear cells（PBMCs）were isolated by lymphocyte stratified fluid and used to isolate single B cell expressing S protein antibody by magnetic beads coupled with S1 protein. Variable region genes of IgG heavy chain and light chain were amplified by nested PCR after reverse transcription of single B cell，which were connected with CMV promoter，IgG leader sequence，IgG constant region and polyA sequence by overlapping PCR to construct antibody linear expression cassette. Linear expression cassette of the heavy chain and light chain from the same B cell was transfected to HEK293T cells to express human monoclonal antibody of SARS⁃CoV⁃2 S protein. Immunoreactivity was detected by immuno⁃ fluorescence while neutralizing activity by pseudovirus neutralization test. Results A total of 26 monoclonal antibodies against SARS⁃CoV⁃2 S protein were expressed，which showed heavy chain and light chain protein bands of IgG antibody at

Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests.

The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops.

Improvement of pest resistance in transgenic tobacco plants expressing dsRNA of an insect-associated gene EcR.

The adoption of pest-resistant transgenic plants to reduce yield loss and pesticide utilization has been successful in the past three decades. Recently, transgenic plant expressing double-stranded RNA (dsRNA) targeting pest genes emerges as a promising strategy for improving pest resistance in crops. The steroid hormone, 20-hydroxyecdysone (20E), predominately controls insect molting via its nuclear receptor complex, EcR-USP. Here we report that pest resistance is improved in transgenic tobacco plants expressing dsRNA of EcR from the cotton bollworm, Helicoverpa armigera, a serious lepidopteran pest for a variety of crops. When H. armigera larvae were fed with the whole transgenic tobacco plants expressing EcR dsRNA, resistance to H. armigera was significantly improved in transgenic plants. Meanwhile, when H. armigera larvae were fed with leaves of transgenic tobacco plants expressing EcR dsRNA, its EcR mRNA level was dramatically decreased causing molting defects and larval lethality. In addition, the transgenic tobacco plants expressing H. armigera EcR dsRNA were also resistant to another lepidopteran pest, the beet armyworm, Spodoptera exigua, due to the high similarity in the nucleotide sequences of their EcR genes. This study provides additional evidence that transgenic plant expressing dsRNA targeting insect-associated genes is able to improve pest resistance.

An agenda-based routing protocol in delay tolerant mobile sensor networks.

Routing in delay tolerant mobile sensor networks (DTMSNs) is challenging due to the networks' intermittent connectivity. Most existing routing protocols for DTMSNs use simplistic random mobility models for algorithm design and performance evaluation. In the real world, however, due to the unique characteristics of human mobility, currently existing random mobility models may not work well in environments where mobile sensor units are carried (such as DTMSNs). Taking a person's social activities into consideration, in this paper, we seek to improve DTMSN routing in terms of social structure and propose an agenda based routing protocol (ARP). In ARP, humans are classified based on their agendas and data transmission is made according to sensor nodes' transmission rankings. The effectiveness of ARP is demonstrated through comprehensive simulation studies.

Fatty acid desaturase-6 (Fad6) is required for salt tolerance in Arabidopsis thaliana.

Fatty acid desaturases play important role in plant responses to abiotic stresses including cold, high temperature, drought, and osmotic stress. In this work, we provide the evidence that Fad6, a chloroplast desaturase, is required for salt tolerance during the early seedling development of Arabidopsis. Expression of Fad6 was responsive to salt and osmotic stress. Compared with the wild-type plants, the fad6 mutant showed reduced tolerance to salt stress, and accumulated more Na(+) and less K(+) under high NaCl stress condition. Furthermore, cellular oxidative damage was more severe in fad6 when treated with high concentrations of NaCl, as indicated by increased electrolyte leakage rate and malondialdehyde production, as well as by decreased activities of anti-oxidative enzymes. All these results suggest that Fad6 is required for salt resistance in Arabidopsis.

Molecular characterization of ThIPK2, an inositol polyphosphate kinase gene homolog from Thellungiella halophila, and its heterologous expression to improve abiotic stress tolerance in Brassica napus.

Inositol polyphosphate kinases play important roles in diverse cellular processes. In this study, the function of an inositol polyphosphate kinase gene homolog named ThIPK2 from a dicotyledonous halophyte Thellungiella halophila was investigated. The deduced translation product (ThIPK2) shares 85% identity with the Arabidopsis inositol polyphosphate kinase AtIPK2beta. Transient expression of ThIPK2-YFP fusion protein in tobacco (Nicotiana tabacum) protoplasts indicates that the protein is localized to the nucleus and plasma membrane, with a minor localization to the cytosol. Heterologous expression of ThIPK2 in ipk2Delta (also known as arg82Delta), a yeast mutant strain that lacks inositol polyphosphate multikinase (Ipk2) activity, rescued the mutant's salt-, osmotic- and temperature-sensitive growth defects. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed ubiquitous expression of ThIPK2 in various tissues, including roots, rosette leaves, cauline leaves, stem, flowers and siliques, and shoot ThIPK2 transcript was strongly induced by NaCl or mannitol in T. halophila as exhibited by real-time PCR analysis. Transgenic expression of ThIPK2 in Brassica napus led to significantly improved salt-, dehydration- and oxidative stress resistance. Furthermore, the transcripts of various stress responsive marker genes increased in ThIPK2 transgenic plants under salt stress condition. These results suggest that ThIPK2 is involved in plant stress responses, and for the first time demonstrate that ThIPK2 could be a useful candidate gene for improving drought and salt tolerance in important crop plants by genetic transformation.

[Effect of Th1/Th2 cytokine immune imbalance on the expression of nerve growth factors in asthma].

OBJECTIVE: To explore the effect of Th1/Th2 cytokines on the expression of nerve growth factor(NGF)in splenic lymphocytes in asthmatic model. METHODS: Four SD rats were sensitized and challenged with ovalbumin to establish an asthmatic model, and the rat splenic lymphocytes were isolated and cultured with ConA. The expressions of NGF mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR), and were observed after the lymphocytes were exogenously added with interferon-gamma(IFN-gamma) or interleukin-4 (IL-4). RESULTS: The lymphocytes of the asthmatic model stimulated by ConA in vitro expressed NGF mRNA in a time-dependent manner. After the lymphocytes had been cultured with IL-4 for 12 h, 24 h, 36 h, and 48 h, 50 microg/L IL-4 upregulated the expressions of NGF mRNA in a time-dependent manner and all the NGF mRNA expressions were significantly higher than the basal values at the same time(all Ps<0.01). After 0, 10, 50, and 100 microg/L IL-4 had been added for 24 h, IL-4 upregulated the expressions of NGF mRNA in a dose-dependent manner and the NGF mRNA expressions were all significantly higher than the values of the lower dose IL-4(all Ps<0.05). After the lymphocytes had been cultured with 10 mug/L IFN-gamma for 0 h, 12 h, 24 h, 36 h, and 48 h, IFN-gamma downregulated the expressions of NGF mRNA in a time-dependent manner and all the NGF mRNA expressions were significantly lower than the basal values at the same time(all Ps<0.01). After 0, 1, 10, and 50 microg/L IFN-gamma have been added for 24 h, IFN-gamma downregulated the expressions of NGF mRNA in a dose-dependent manner and all the NGF mRNA expressions were significantly lower than the values of the lower IFN-gamma dose(all Ps<0.05). CONCLUSION: In the splenic lymphocytes of asthmatic rats, IL-4, one of the Th2 cytokines, can upregulate the expressions of NGF; IFN-gamma, one of the Th1 cytokines, can downregulate the expressions of NGF both in a time-dependent manner and in a dose-dependent manner. Th1/Th2 cytokine immune imbalance may indirectly induce the airway neurogenic inflammation by regulating the NGF mRNA expression.

[Experimental study on the mechanisms of regulating airway neurogenic inflammation in asthma by never growth factor and leukemia inhibitory factor].

OBJECTIVE: To study the mechanisms of regulating airway neurogenic inflammation in asthma by never growth factor (NGF) and leukemia inhibitory factor (LIF), and then to explore new targets in treating asthma. METHODS: Adult male SD rats (n 36) were divided into the normal group, the asthmatic group and the anti-NGF group at random. There were 12 rats in each group. The asthma models were established by sensitization and challenge with ovalbumin, and the asthma model was treated with anti-NGF. The expression of NGF, LIF and substance P (SP) in lung tissue or in doral root ganglion of each rat were detected by immunohistochemistry and hybridisation in situ. RESULTS: (1) The gray-levels of NGF protein/NGF mRNA, LIF protein/LIF mRNA in the lungs were 157 +/- 7, 138 +/- 8, 156 +/- 6, 141 +/- 10 for the asthmatic group respectively, 183 +/- 7, 190 +/- 7, 187 +/- 7, 181 +/- 8 for the normal control group respectively, and 177 +/- 6, 169 +/- 9, 178 +/- 7, 172 +/- 9 for the asthmatic group with anti-NGF treatment. There were significant differences in gray-level of NGF protein/NGF mRNA, LIF protein/LIF mRNA among those three groups (t = 19.40, 15.80, 20.38, [corrected] 14.79, all P < 0.01). (2) The gray-levels of NGF protein/LIF protein, SP protein/SP mRNA in the doral root ganglions were 136 +/- 8, 148 +/- 6, 140 +/- 8, 128 +/- 8 for the asthmatic group respectively, 185 +/- 7, 187 +/- 8, 174 +/- 7, 180 +/- 8 for the normal control group respectively, and 164 +/- 6, 170 +/- 8, 163 +/- 9, 157 +/- 7 for the asthmatic group with anti-NGF treatment. There were also significant differences in gray-level of NGF protein/LIF protein, SP protein/SP mRNA among those three groups (t = 29.50, 22.65, 23.12, 28.71, all P < 0.01). CONCLUSION: Enhancing the synthesis and release of SP in doral root ganglion may be one of the mechanisms by which NGF and LIF regulate airway neurogenic inflammation in asthmatic rats, and this mechanism can be depressed by the intervention of anti-NGF.

[Regulation effect of nerve growth factor on Ras-MAPK signal transduction pathway in neurogenic inflammation of asthma].

OBJECTIVE: To investigate the regulatory effect of nerve growth factor (NGF) on Ras-MAPK signal transduction pathway in neurogenic inflammation of asthma. METHODS: Thirty-six Sprague-Dawley rats were randomly divided into 3 groups (control group, asthma group and anti-NGF group). The asthmatic model was established by ovalbumin inhalation and injection. The protein expressions of pan-Ras, pERK and c-fos in the dorsal root ganglion and lung of the asthma group and the control group were examined by immunohischemical method. Anti-NGF antibody was used to investigate how it affected the protein expression of pan-Ras, pERK and c-fos in the dorsal root ganglion and the lung of the asthma group. PD98059 (the inhibitor of MAPK) and PMA (the enhancer of PKC) were used to culture the NHBEC. Cell extracts were analyzed for pERK, total-ERK and c-fos by Western blot. RESULTS: The protein expressions of pan-Ras, pERK and c-fos in the lung and dorsal root ganglion of the asthma group were significantly higher than those of the control group (P < 0.01). The protein expressions of pan-Ras, pERK and c-fos were decreased by the anti-NGF treatment (P < 0.01) . The expressions of epithelial pERK and c-fos in the NGF group were significantly higher than those in the control group, and PD98059 could inhibit NGF inducing NHBEC to produce pERK and c-fos. PMA could enhance the effects of NGF. CONCLUSION: NGF may play a role in the pathogenesis of neurogenic inflammation in asthma through Ras-MAPK signal transduction pathway.

LIF upregulates expression of NK-1R in NHBE cells.

Leukemia inhibitory factor (LIF), a cytokine at the interface between neurobiology and immunology, is mainly mediated through JAK/STAT pathway and MAPK/ERK pathway. Evidence suggested LIF is related to the higher expression of neurokinin-1 receptor (NK-1R) in asthma. In this study, the immunohistochemistry stain showed the expressions of NK-1R, LIF, p-STAT3, and p-ERK1/2 in the lung tissues of allergic rats were increased compared with the controls, and the main positive cell type was airway epithelial cell. Normal human bronchial epithelial cells were treated with LIF in the presence or absence of AG490 (JAK2 inhibitor), PD98059 (MEK inhibitor), and the siRNA against STAT3. Western blot and RT-PCR indicated that LIF induced the expression of NK-1R, which was inhibited by the inhibitors mentioned above. No significant interaction was found between JAK/STAT pathway and MAPK/ERK pathway. In summary, bronchial epithelial cell changes in asthma are induced by LIF which promotes the expression of NK-1R, and JAK/STAT pathway and MAPK/ERK pathway may participate in this process.

[Changes of nerve growth factor and its receptors in the lung tissues in asthmatic rats and their effects on the airway inflammation].

OBJECTIVE: To determine the expression of nerve growth factor (NGF), tyrosine kinase receptor A (trkA), and pan-neurotrophin receptor (p75) in the lung tissues in asthmatic rats, and to explore their effects on the airway inflammation. METHODS: Thirty-two SD rats were randomly divided into 4 groups: the control, asthma, NGF and anti-NGF groups. The asthmatic model was established by the inhalation and injection of ovalbumin. The total cell count and differential cell count in the bronchoalveolar lavage fluid (BALF) were performed. The pathologic changes in the lung tissues of the 4 groups was detected by HE staining. The NGF mRNA expression in the lung tissues of the asthma and control groups was determined by reverse transcription-polymerase chain reaction (RT-PCR). The changes of trkA and p75 mRNA expressions in the lung tissues in the 4 groups were also investigated by RT-PCR. RESULTS: Compared with the control group, the BALF total cell, the BALF eosinophils (Eos), and the BALF lymphocytes (Lyms) significantly increased (All P <0. 001) in the asthma group; and the lung tissues of the asthma group had more infiltrating inflammatory cells. Not only the expression of NGF mRNA, but also its receptors trkA and p75 mRNA in the lung tissues were significantly higher in the asthma group than those in the control group (All P < 0.01). Positive correlation was found between the expression of NGF mRNA and the BALF total cell, the BALF Lyms in the asthma group. Compared with the asthma group, the total cell, the Eos, and the lyms in BALF in the NGF group significantly increased (All P < 0.01), and the lungs of the NGF group had apparent inflammatory changes. The expre-ssions of p75 and trkA mRNA were enhanced significantly (All P < 0.05). Compared with the asthma group, the total cell, the Eos, and the lyms in BALF in the anti-NGF group significantly decreased (All P < 0.001), and the lungs of the anti-NGF group showed alleviative inflammatory changes. The expre-ssions of p75 and trkA mRNA significantly decreased (All P < 0.01). CONCLUSION: In lungs of asthmatic rats, the elevated expression of NGF mRNA is closely related to the airway inflammation. NGF can upregulate the expressions of p75 and trkA mRNA in asthmatic rats, and then may promote their role in the airway neuronal inflammation in asthma.