A MAP kinase cascade broadly regulates the lifestyle of Sclerotinia sclerotiorum and can be targeted by HIGS for disease control.

Sclerotinia sclerotiorum causes white mold or stem rot in a wide range of economically important plants, bringing significant yield losses worldwide. Control of this pathogen is difficult as its resting structure sclerotia can survive in soil for years, and no Resistance genes have been identified in S. sclerotiorum hosts. Host-induced gene silencing (HIGS) has shown promising effects in controlling many fungal pathogens, including S. sclerotiorum. However, better molecular genetic understanding of signaling pathways involved in its development and pathogenicity is needed to provide effective HIGS gene targets. Here, by employing a forward genetic screen, we characterized an evolutionarily conserved mitogen-activated protein kinase (MAPK) cascade in S. sclerotiorum, consisting of SsSte50-SsSte11-SsSte7-Smk1, which controls mycelial growth, sclerotia development, compound appressoria formation, virulence, and hyphal fusion. Moreover, disruption of the putative downstream transcription factor SsSte12 led to normal sclerotia but deformed appressoria and attenuated host penetration, as well as impaired apothecia formation, suggestive of diverged regulation downstream of the MAPK cascade. Most importantly, targeting SsSte50 using host-expressed double-stranded RNA resulted in largely reduced virulence of S. sclerotiorum on both Nicotiana benthamiana leaves and transgenic Arabidopsis thaliana plants. Therefore, this MAPK signaling cascade is generally needed for its growth, development, and pathogenesis and can serve as ideal HIGS targets for mitigating economic damages caused by S. sclerotiorum infection.

Preparation of monoclonal antibodies against Epstein-Barr virus glycoprotein 350.

Epstein-Barr virus (EBV) is the first identified human oncogenic herpesvirus infecting over 90% of the adults worldwide. However, the safe and effective prophylactic vaccine has not been licensed. The major glycoprotein 350 (gp350) on the EBV envelope is the main target for neutralizing antibodies, and gp350 (aa15-320) was used for the development of monoclonal antibodies in present study. The purified recombinant gp35015-320aa with an estimated molecular weight of 50 kDa was used to immunize six-week-old BALB/c mice, and the hybridoma cell lines that stably secreted monoclonal antibodies (mAbs) were obtained. The ability of developed mAbs for capturing and neutralizing EBV was evaluated, and mAb 4E1 presented better performance to block the infection of EBV in cell line Hone-1. The mAb 4E1 recognized the epitope. Its sequence of variable region genes (VH and VL) presented a unique identity which hadn't been reported. The developed mAbs might benefit the antiviral therapy and immunologic diagnosis for EBV infection.

Magnetic particle-based chemiluminescence immunoassay for serum human heart-type fatty acid binding protein measurement.

OBJECTIVES: Human heart-type fatty acid binding protein (HFABP) is a biomarker for diagnosis, risk assessment, and prognosis of acute myocardial infarction, and we aimed to establish an immunoassay for HFABP quantitation. METHODS: Human HFABP monoclonal antibodies (mAbs) were developed, evaluated by enzyme-linked immunosorbent assay, and a chemiluminescence enzyme immunoassay (CLEIA) generated. Analytical performance of the CLEIA was evaluated by measuring serum HFABP. RESULTS: The prokaryotically expressed rHFABP was purified and four anti-HFABP mAbs with superior detection performance were obtained after immunizing BALB/c mice. MAbs 2B8 and 6B3 were selected as respective capture and detection antibodies for HFABP measurement by CLEIA (detection range, 0.01-128 µg/L). Results using the CLEIA showed excellent correlation (r, 0.9622) and the correlation coefficient was 0.9809 (P < 0.05) by the Tukey test statistical analysis with those of latex-enhanced immunoturbidimetry in hospitals. CONCLUSION: Our mAbs and CLEIA for HFABP detection represent new diagnostic tools for measurement of human serum HFABP.

SsCak1 Regulates Growth and Pathogenicity in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating fungal pathogen that causes severe crop losses worldwide. It is of vital importance to understand its pathogenic mechanism for disease control. Through a forward genetic screen combined with next-generation sequencing, a putative protein kinase, SsCak1, was found to be involved in the growth and pathogenicity of S. sclerotiorum. Knockout and complementation experiments confirmed that deletions in SsCak1 caused defects in mycelium and sclerotia development, as well as appressoria formation and host penetration, leading to complete loss of virulence. These findings suggest that SsCak1 is essential for the growth, development, and pathogenicity of S. sclerotiorum. Therefore, SsCak1 could serve as a potential target for the control of S. sclerotiorum infection through host-induced gene silencing (HIGS), which could increase crop resistance to the pathogen.

A Forward Genetic Screen in Sclerotinia sclerotiorum Revealed the Transcriptional Regulation of Its Sclerotial Melanization Pathway.

Most plant fungal pathogens that cause worldwide crop losses are understudied, due to various technical challenges. With the increasing availability of sequenced whole genomes of these non-model fungi, effective genetic analysis methods are highly desirable. Here, we describe a newly developed pipeline, which combines forward genetic screening with high-throughput next-generation sequencing to enable quick gene discovery. We applied this pipeline in the notorious soilborne phytopathogen Sclerotinia sclerotiorum and identified 32 mutants with various developmental and growth deficiencies. Detailed molecular studies of three melanization-deficient mutants provide a proof of concept for the effectiveness of our method. A master transcription factor was found to regulate melanization of sclerotia through the DHN (1,8-dihydroxynaphthalene) melanin biosynthesis pathway. In addition, these mutants revealed that sclerotial melanization is important for sclerotia survival under abiotic stresses, sclerotial surface structure, and sexual reproduction. Foreseeably, this pipeline can be applied to facilitate efficient in-depth studies of other non-model fungal species in the future.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Bufotalin-loaded biomimetic Prussian blue nanoparticles for colorectal cancer chemo-photothermal ferroptosis therapy.

Purpose: We constructed biomimetic nanoparticles with biocompatible, tumor-targeting, laser-responsive properties for ferroptosis-induced colorectal cancer chemo-photothermal therapy, with the aim to realize double-hit ferroptosis treatment for colorectal cancer. Methods: The nanoparticles were prepared by first loading the chemotherapy drug bufotalin (CS-5) with Prussian blue (PB), then combining a hybridized erythrocyte-tumor membrane (M) with PB@CS-5 to produce PB@CS-5@M. The chemo-photothermal therapy efficiency of PB@CS-5@M was tested by in vitro and in vivo experiments. Results and conclusion: The combined PB and CS-5 act as promising ferroptosis inducers to enhance ferroptosis efficacy. The hyperthermia induced by laser stimulation can trigger PB to release CS-5 and iron and ferrous ions, which further promotes ferroptosis.

A cAMP phosphodiesterase is essential for sclerotia formation and virulence in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a plant pathogenic fungus that causes white mold or stem rot diseases. It affects mostly dicotyledonous crops, resulting in significant economic losses worldwide. Sclerotia formation is a special feature of S. sclerotiorum, allowing its survival in soil for extended periods and facilitates the spread of the pathogen. However, the detailed molecular mechanisms of how sclerotia are formed and how virulence is achieved in S. sclerotiorum are not fully understood. Here, we report the identification of a mutant that cannot form sclerotia using a forward genetics approach. Next-generation sequencing of the mutant's whole genome revealed candidate genes. Through knockout experiments, the causal gene was found to encode a cAMP phosphodiesterase (SsPDE2). From mutant phenotypic examinations, we found that SsPDE2 plays essential roles not only in sclerotia formation, but also in the regulation of oxalic acid accumulation, infection cushion functionality and virulence. Downregulation of SsSMK1 transcripts in Sspde2 mutants revealed that these morphological defects are likely caused by cAMP-dependent inhibition of MAPK signaling. Moreover, when we introduced HIGS construct targeting SsPDE2 in Nicotiana benthamiana, largely compromised virulence was observed against S. sclerotiorum. Taken together, SsPDE2 is indispensable for key biological processes of S. sclerotiorum and can potentially serve as a HIGS target to control stem rot in the field.

Supplementing a specific synbiotic suppressed the incidence of AOM/DSS-induced colorectal cancer in mice.

In this study, we evaluated the effect of a specific synbiotic on CAC (AOM/DSS-induced colitis-associated cancer). We confirmed that the synbiotic intervention was able to protect the intestinal barrier and inhibit CAC occurrence via upregulating tight junction proteins and anti-inflammatory cytokines, and downregulating pro-inflammatory cytokines. Moreover, the synbiotic significantly improved the disorder of the colonic microbiota of CAC mice, promoted the formation of SCFAs and the production of secondary bile acids, and alleviated the accumulation of primary bile acids in the CAC mice. Meanwhile, the synbiotic could significantly inhibit the abnormal activation of the intestinal Wnt/ß-catenin signaling pathway significantly related to IL-23. In a word, the synbiotic can inhibit the occurrence and development of colorectal tumors and it may be a functional food to prevent inflammation-related colon tumors, and the research also provided a theoretical basis for improving the intestinal microecological environment through diet therapy.

Characterization of genotype IV hepatitis E virus-like particles expressed in E.coli.

HEV (Hepatitis E virus) is an infectious disease transmitted between humans and animals, which poses a severe threat to the biological safety and property throughout the world. The disease is especially severe in patients with potential liver cirrhosis and women during pregnancy. There is no specific and thorough HEV treatment at present. The development of hepatitis E virus vaccine is vital to the prevention of viral hepatitis worldwide. Since HEV cannot grow adequately in vitro, vaccine developed by devitalized virus particles does not work. Exploration of HEV-like structures is essential for the development of functional vaccines against HEV infection. ORF2 encodes the structural proteins of HEV, some of which can automatically assemble into virus-like particles (VLP) in this experiment, the recombinant capsid protein p27 was expressed in E. coli and the VLP formed by p27 was used to immunize mice. The results showed that the VLP formed by recombinant P27 had similar particle size to that of HEV; the immune dose produced by p27 was positively correlated with the immune effect. Compared with other genetic engineering subunit vaccines, P27 protein has a better application prospect.

Development of magnetic particle-based chemiluminescence immunoassay for measurement of SARS-CoV-2 nucleocapsid protein.

BACKGROUND: Recently, the Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 infection has spread rapidly around the world, becoming a new global pandemic disease. Nucleic acid detection is the primary method for clinical diagnosis of SARS-CoV-2 infection, with the addition of antibody and antigen detection. Nucleocapsid protein (NP) is a kind of conservative structural protein with abundant expression during SARS-CoV-2 infection, which makes it an ideal target for immunoassay. METHODS: The coding sequence for SARS-CoV-2-NP was obtained by chemical synthesis, and then inserted into pET28a(+). The soluble recombinant NP (rNP) with an estimated molecular weight of 49.4 kDa was expressed in E. coli cells after IPTG induction. Six-week-old BALB/c mice were immunized with rNP, and then their spleen cells were fused with SP2/0 cells, to develop hybridoma cell lines that stably secreted monoclonal antibodies (mAbs) against NP. The mAbs were preliminarily evaluated by enzyme-linked immunosorbent assay (ELISA), and then used to develop a magnetic particle-based chemiluminescence enzyme immunoassay (CLEIA) for measurement of SARS-CoV-2-NP. RESULTS: mAb 15B1 and mAb 18G10 were selected as capture and detection antibody respectively to develop CLEIA, due to the highest sensitivity for rNP detection. The proposed CLEIA presented a good linearity for rNP detection at a working range from 0.1 to 160 µg/L, with a precision coefficient of variance below 10 %. CONCLUSION: The newly developed mAbs and CLEIA can serve as potential diagnostic tools for clinical measurement of SARS-CoV-2-NP.

Fructooligosaccharide supplementation alleviated the pathological immune response and prevented the impairment of intestinal barrier in DSS-induced acute colitis mice.

The dysbiosis of gut microbiota is closely related to the occurrence and development of inflammatory bowel disease (IBD). The manipulation of intestinal flora through prebiotics or probiotics is expected to induce and maintain the remission of IBD symptoms. 6-week-old C57BL/J mice were daily gavaged with fructooligosaccharides (FOS) or the synbiotic two weeks before the administration of dextran sulfate sodium (DSS). The supplementation of FOS or synbiotic could significantly ameliorate the body weight loss and colon histological damage in DSS-induced acute colitis mice. The altered composition of gut microbiota in acute colitis mice was reversed by FOS or Synbiotic supplementation, with a characteristic of decreased abundance of Mucispirillum. Both FOS and synbiotic mitigated DSS-induced loss of mucus protein (MUC2) and epithelium tight junction proteins (ZO-1, Occluding, Claudin1) in colon mucosa. The expression of pro-inflammatory cytokines (IL-6 and TNF-α) was decreased by FOS or synbiotic treatment, while the expression of Tbx21 and IL-10 was increased. The results suggested that the modulation of gut microbiota by FOS or synbiotic supplementation could decrease the inflammation potential of colonized commensals, which prevented the impairment of the intestinal barrier and induced a regulation of immune response in DSS-induced acute colitis mice.

Development of magnetic particle-based chemiluminescence immunoassay for measurement of human procalcitonin in serum.

BACKGROUND: Serum procalcitonin (PCT) has been recognized as a primary biomarker in bacterial infections, and monitoring its concentration could help to evaluate the prognosis of sepsis and guide the antibiotic administration. We aimed to establish a fast and accurate immunoassay for PCT quantitation. METHODS: Our newly developed monoclonal antibodies (mAbs) against human PCT were preliminarily evaluated by enzyme-linked immunosorbent assay and then used to develop a chemiluminescence enzyme immunoassay (CLEIA). The proposed CLEIA was assessed in analytical performance and applied to measurement of serum PCT. RESULTS: mAb 2D3 and mAb 8F6 were selected as capture and detection antibody respectively, due to the highest sensitivity for PCT detection with no cross reaction to calcitonin gene-related peptides. The proposed CLEIA based on mAb pair of 2D3/8F6-AP was characterized for a working range from 0.03 to 100 µg/L. An excellent correlation was observed between our proposed assay and the VIDAS BRAHMS PCT assay (r: 0.9825). CONCLUSION: Our newly developed mAbs and CLEIA can serve as important diagnostic tools for measurement of human PCT in serum.

Analysis of the meiotic transcriptome reveals the genes related to the regulation of pollen abortion in cytoplasmic male-sterile pepper (Capsicum annuum L.).

CMS, which refers to the inability to generate functional pollen grains while still producing a normal gynoecium, has been widely used for pepper hybrid seed production. Pepper line 8214A is an excellent CMS line exhibiting 100% male sterility and superior economic characteristics. A TUNEL assay revealed the nuclear DNA is damaged in 8214A PMCs during meiosis. TEM images indicated that the 8214A PMCs exhibited asynchronous meiosis after prophase I, and some PMCs degraded prematurely with morphological features typical of PCD. Additionally, at the end of meiosis, the 8214A PMCs formed abnormal non-tetrahedral tetrads that degraded in situ. To identify the genes involved in the pollen abortion of line 8214A, the transcriptional profiles of the 8214A and the 8214B anthers (i.e., from the fertile maintainer line) during meiosis were analyzed using an RNA-seq approach. A total of 1355 genes were determined to be differentially expressed, including 424 and 931 up- and down- regulated genes, respectively, in the 8214A anthers during meiosis relative to the expression levels in the 8214B. The expression levels of ubiquitin ligase and cell cycle-related genes were apparently down-regulated, while the expression of methyltransferase genes was up-regulated in the 8214A anthers during meiosis, which likely contributed to the PCD of these PMCs during meiosis. Thus, our results may be useful for revealing the molecular mechanism regulating the pollen abortion of CMS pepper.

[The distribution of calcium in the stigma and style of tobacco during pollen germination and tube growth].

After pollen grains of tobacco landed on stigma they begin to hydrate and form many small vesicles containing some calcium grains in cytoplasm. The calcium stored in pollen wall is released into tectum of stigma to make a calcium-rich environment. When a pollen tube penetrates the tectum and grows between stigma cells, numerous calcium precipitates appear in the tip tube wall. The length of style of tobacco is 4 cm, and the pollen tube need take 44 h to reach the ovary. The style was artificially divided into 4 stages and each 1 cm respectively. There were only a few of calcium precipitates in the transmitting tissue of style from anthesis to 11 h after pollination. A calcium gradient in the transmitting tissue of style was formed at 22 h after pollination: only a few calcium precipitates found in the transmitting tissue of the style under stigma and at stage 1, 2 and 3, and many of them were located in the transmitting tissue of style near ovary (stage 4). When the flowers were emasculated and unpollinated at 1 d after anthesis, no calcium gradient in the transmitting tissue of style could be identified because some precipitates were also accumulated in the transmitting tissue at stage 1. When a flower without pollination was kept for 3 d, some calcium precipitates were formed in the cells of stigma, and the cells of the whole transmitting tissue contained the same quantity of calcium precipitates. To check the ability of pollen to germinate and grow in a low calcium environment, pollen grains were cultured in a medium containing 0-0.1% CaCl(2).2H(2)O. The result of in vitro assay confirmed that tobacco pollen can germinate and the pollen tube can grow in an environment with a very low concentration of calcium, which may be similar to the environment in the stigma. A few calcium precipitates were accumulated in stigma and upper transmitting tissue of tobacco to make a calcium gradient in the style. If the calcium in the style at 1 cm increases it will be increased more at 4 cm, and more in ovules, and more in synergid cells to keep the calcium gradient. When the emasculated flowers were not pollinated for 3 days the calcium in upper transmitting tissue evidently increases. The calcium in style is abundant in all plants, but the distribution of calcium in style is different between different plant species. For this difference, it may differ from types of style, and in the plants with short style the calcium gradient in the style is too small to be detected. But for tobacco with style 4 cm long, the gradient can be identified using antimonate method.

[Calcium distribution in fertile and sterile anthers of a genic male sterile Chinese cabbage].

Potassium antimonite was used to locate calcium in the fertile and sterile anthers of a genic male sterile Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) to probe the relation between Ca(2+) and fertility and sterility of anthers of the cabbage. During fertile anther development, calcium granules increase in number in anther wall cells after meiosis, and then appeared also in locule, suggesting a calcium influx into locule from anther wall cells (Plate I-4). Then the number of calcium granules in microspore cytoplasm also increased at early stage (Plate II-1), accumulated mainly on the membrane of small vacuoles which were fusing to form big ones to make a polarity in the cell and to prepare asymmetric division of microspore (Plate II-3,4). After microspore division and the big vacuole decomposition, many calcium granules accumulated again on the membrane of the vacuoles (Plate III-1,2), displaying calcium regulates vacuole formation and decomposition during pollen development. In sterile anthers, abnormal distribution of calcium granules first appeared in callus wall of microspore mother cell (Plate IV-1). However, only a few calcium granules appeared in early microspores, which then could not form small vacuoles and finally a big vacuole (Plate IV-2,3). The aborting microspores degenerate by cytoplasm shrinking (Plate IV-5,6). The difference pattern of distribution of calcium granules between the fertile and sterile anthers indicates that anomalies in the distribution of calcium accumulation are correlated with the failure of pollen development and pollen abortion.

[The dynamic of calcium distribution during megasporegenesis of lettuce (Lactuca sativa L.)].

Potassium antimonite was used to deposit calcium in the young ovule of lettuce (Lactuca sativa L.) at megasporogenesis stage to study the relationship between calcium and megaspore degeneration. At the megaspore mother cell stage, few calcium granules were formed in the cell (Plate I-1, 2). After meiosis of megaspore mother cell and forming an arrayed tetrad in a line (Plate I-3), three megaspores degenerated one by one from the micropyle end. In the process of degeneration, the numbers of calcium granules decreased in the three megaspores. After the first megaspore degenerated, the number of calcium granules decreased in the second megaspore, which began to degenerate (Plate II-7, 8). The third megaspore also had its number of calcium granules diminishing before it degenerated (Plate III-13, 14). The fourth megaspore always accumulated many calcium granules in the cytoplasm during its development (Plate IV-17, 18) and finally becomes functional one that will develop into an embryo sac (Plate IV-20). Megaspore degeneration is a process of programmed cell death which may be closely related with change in calcium content: when a megaspore of tetrad decreases calcium content the cell begins to degenerate, and when calcium increases in the cell, it will continue to develop into a functional megaspore. This is the first report about calcium distribution in megaspores of a tetrad during megasporogenesis in higher plants and will open a door to study the physiological function of calcium in megasporogenesis.

[Prokaryotic expression and characterization of truncated mutant capsid protein of genotype IV hepatitis E virus].

A truncated mutant of the Open Reading Frame 2 (ORF2, aa384-606) was amplified from cDNA of genotype IV hepatitis E virus (HEV) by polymerase chain reaction (PCR), subcloned to expression plasmid pTO-T7, and expressed in Escherichia coli. SDS-PAGE and Western blotting were used to detect and identify the recombinant protein, namely rP24. After washing of inclusion bodies, dissolving in denaturing agents, refoldeding by dialysis, ion exchange chromatography and gel chromatography, dynamic light scatter was used to study the hydrated radius of rP24. Western blotting was applied to detect the immunoreactivity of rP24, and mouse immunity test and indirect enzyme linked immunosorbent assay (ELISA) were applied to evaluate the immunogenicity and the detection rate of HEV positive and negative serum. SDS-PAGE and Western blotting show that rP24 was highly expressed in the form of inclusion bodies after induction, and had strong immunoreactivity to monoclonal antibody (McAb) 15B2. After a multi-step purification of rP24, Western blotting indicated that the purified rP24 also had strong immunoreactivity to neutralizing McAb 8C11 and HEV positive serum, suggesting that rP24 simulated the nature structure of HEV capsid protein. Dynamic light scatter demonstrated that the average hydration radius of purified rP24 was 7.48 nm. The mouse immunity test showed that the purified rP24 also had good immunogenicity, and the period of serum antibodies converted from negative to positive was very short, but the antibodies maintained more than 20 weeks. Indirect ELISA tests showed that the detection rate of was the same as anti-HEV-IgG diagnostic kit (Wan Tai corporation). Taken together, the rP24 simulated the neutralizing epitopes of natural HEV, and had strong immunoreactivity and immunogenicity. It provided a basis for the further investigation of the difference of infection mechanism between genotype I and genotype IV HEV.

Expression, purification, and characterization of hepatitis B virus surface antigens (HBsAg) in yeast Pichia Pastoris.

Prevention of the prevalence of HB depends upon the development of efficient diagnostic reagent and preventive vaccine. Pichia pastoris offers many advantages over the other expression systems in the production of recombinant HBsAg. In this study, we reported that the recombinant P. pastoris strains were cultured in shake flasks and then scaled up in a 5.0-l bioreactor: approximately 27 mg/l of the protein and the maximal cell OD at 600 nm of 310 were achieved in the bioreactor. The recombinant HBsAg was purified by three steps of purification procedures. SDS-PAGE showed that the purified recombinant HBsAg constituted only one homogeneous band of approximately 24 kDa. CsCl density gradient ultracentrifugation assay indicated that the density of the HBsAg was 1.2 mg/ml, which was in agreement with the natural HBsAg, the HBsAg expressed in Saccharomyces cerevisiae and in mammalian cells. Electron microscope observation revealed that the purified recombinant HBsAg was homogeneous 22-nm particles, suggesting the HBsAg expressed in P. pastoris was self-assembled to virus-like structures. Competitive ELISA indicated that P. pastoris-derived HBsAg possessed the excellent immunoreaction with anti-HBsAg. Animal immunization showed that the immunogenicity of P. pastoris-derived HBsAg was superior to that of S. cerevisiae-derived HBsAg. Together, our results demonstrated that the recombinant HBsAg expressed in P. pastoris could provide promising, inexpensive, and large-scale materials for the diagnostic reagent and vaccine to prevent HBV infection.

Optimization of fermentation conditions for maximal recombinant hepatitis B surface antigen particle production in Pichia pastoris.

Hepatitis B virus (HBV) infection can cause the severe threat to the health of the people around the world. It depends upon the development of efficient diagnostic reagent and vaccine to prevent the prevalence of HB. In this study, we constructed the high expression recombinant Pichia pastoris and performed the screening tests in shake flasks to obtain the optimal values of several key fermentation parameters. Based on their effects on the growth and expression level of recombinant strains, FBS was the optimal industrial medium. The optimal values for the dissolve oxygen (DO), the final concentrations of methanol and the pH values were 50 mL, 1% (V/V) and 5.4-6.0, respectively. The optimal values of the parameters simulated in shake flasks were successfully scaled up to 10 L bioreactors to achieve high-throughput production: 310 OD600 in biomass and 27 mg/L in recombinant HBsAg. The expressed recombinant HBsAg in P. Pastoris was confirmed by SDS-PAGE and Western blotting. Electron microscopy examination showed that the purified protein could be self-assembled to 22 nm virus-like particles. The results provided a basis for industrial scale-up production of diagnostic reagent and vaccine of next generation against HB.

[Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity].

The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.