Kuohai Fan

The cytological observation of immune adherence of porcine erythrocyte.

Abstract The immune adherence (IA) between the porcine erythrocytes and the opsonized Escherichia coli carried green fluorescent protein gene (GFP-E.coli) were detected by the fluorescence microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) with an attempt to verify the existence of IA between the porcine erythrocytes and complemented-opsonized microbes. Under fluorescence microscopy, GFP-E.coli opsonized by fresh rabbit serum complement adhered to the erythrocytes and could not be detached by PBS washing, and no IA was observed between the erythrocytes and nonopsonized GFP-E.coli after co-incubation. SEM and TEM also revealed the existence of IA between the serum complement-opsonized GFP-E.coli membrane and the erythrocyte membrane. The partial complement receptor type 1 (CR1)-like gene from porcine was generated by RT-PCR and rapid amplification of cDNA 3’ end (3’ RACE) (157bp and 578bp), both of which have high similarity with published mammals CR1 gene. The sequences were spliced based on homology comparison and submitted to GenBank (GenBank Accession No. JX033989). These results indicated that the porcine erythrocytes were able to bind to the opsonized microorganisms. Furthermore, the sequencing results confirmed that the CR1-like gene exists in porcine.

Expression and Purification of Soluble Porcine Cystatin 11 in Pichia pastoris

Cystatin 11 (CST11) belongs to the cystatin type 2 family of cysteine protease inhibitors and exhibits antimicrobial activity in vitro. In this study, we describe the expression and purification of recombinant porcine CST11 in the Pichia pastoris system. We then assess its antimicrobial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis by liquid growth inhibition assay. Kinetic studies indicate that the recombinant porcine CST11 has high potency against E. coli and S. aureus. Scanning electronic microscope analysis showed that CST11 might be targeting the bacterial membrane and, thus, could potentially be developed as a therapeutic agent for inhibiting microbe infection without the risk of antibiotic resistance.

Cloning and bioinformatics analysis of a full-length cDNA of porcine CR1-like gene

Jing Cheng1, Junbing Jiang1, Junxing Zhao1, Zhirui Wang2,3, Yaogui Sun1, Haili Ma1, Kuohai Fan1, Wei Yin1, Na Sun1, Zhiwei Wang1, Xin Zhao1, and Hongquan Li1* College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02148, USA MGH-DF/HCC Recombinant Protein Expression and Purification Core, Boston, MA 02148, USA *Correspondence address. Tel/Fax: þ86-354-6288409; E-mail: livets@163.com

Expression and purification of the recombinant porcine NK-lysin in Pichia pastoris and observation of anticancer activity in vitro

ABSTRACT Natural killer (NK)-lysin, a broad-spectrum antimicrobial peptide, has antitumor and antibactericidal activities against both gram-positive and gram-negative bacteria. In this study the recombinant porcine NK-lysin was expressed and purified in the Pichia pastoris system, and then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to assess its anticancer activity in vitro. The results showed that the recombinant porcine NK-lysin possesses potent antitumor activity against the human hepatocellular carcinoma cell line SMMC-7721 in a time- and dose-dependent manner, but has negligible hemolysis activity against human erythrocytes. Scanning electronic microscopy was used to directly observe the ultrastructure of SMMC-7721 cells treated with NK-lysin; untreated cells showed lamellipodia and filopodia scattered with the cell surface, with good cell–cell contacts among neighboring cells. In contrast, treated tumor cells exhibited marked alterations in cell morphology, and cell–cell contacts disappeared among neighboring cells. Compared with the untreated tumor cells, the tumor cells treated with NK-lysin for 12 and 24 hr were suppressed for the expression of fascin 1. Thus, the recombinant porcine NK-lysin potentially could be developed as a therapeutic agent for inhibiting tumor growth.

The immune adherence receptor CR1-like existed on porcine erythrocytes membrane

In the present study, we obtain a mouse anti-porcine complement receptor type 1 (CR1)-like monoclonal antibody (McAb) and use this McAb to verify the existence of CR1-like protein on porcine erythrocytes. Our results confirm that CR1-like protein is localized on the surface of porcine erythrocytes. Mouse immunoglobulin G inhibited the binding of serum-opsonized green fluorescent protein-expressing Escherichia coli to porcine erythrocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicates that CR1-like McAb reacts with biochemically-purified porcine erythrocyte membrane fractions, with a clear band at 135 kDa to 140 kDa. We postulate that the 135 kDa to 140 kDa membrane protein is the equivalent of the porcine erythrocyte CR1-like protein.

Differences in expression density and molecular weight of CR1-Like in erythrocytes of landrace swine

Discussion. CR1-like was dispersed on the surface of porcine erythrocytes and promoted immune adherence. There have been no reports on whether differences in the expression levels and/or molecular weights of CR1-like in erythrocytes represent diversity in different individuals, and if so, whether this diversity influences the immune adherence of erythrocytes and/or whether the diversity is associated with CR1-like polymorphisms. At present, five candidate genes that are related to the differences above were found. Research examining erythrocyte immune adherence and CR1-like genes is under way. These results will provide theoretical data for future studies of the immunological mechanism of CR1-like in porcine erythrocytes.

Suppression of the Wnt signaling pathway may contribute to the inhibition of proliferation of human hepatocellular carcinoma SMMC-7721 cells by esculetin

The aims of the present study were to investigate the effects of esculetin on the proliferation of human hepatocellular carcinoma SMMC-7721 cells and to determine the underlying mechanism behind this activity. An MTT assay was used to assess cell proliferation, reverse transcription-quantitative polymerase chain reaction was used to determine the relative mRNA expression levels of β-catenin, c-Myc and cyclin D1, and western blot analysis was utilized to determine the levels of the associated proteins. Compared with the dimethyl sulfoxide control, esculetin reduced the cell viability of SMMC-7721 and HL-7702 cells in a dose- and time-dependent manner. Treatment of SMMC-7721 cells with esculetin resulted in downregulation of the mRNA and protein levels of β-catenin, c-Myc and cyclin D1. Esculetin increased the phosphorylation of β-catenin at Ser33/Ser37/Thr41 and inhibited the proliferation of human hepatoma SMMC-7721 cells by suppressing the Wnt signaling pathway. The results of the present study suggest that esculetin inhibited the Wnt/β-catenin signaling pathway in SMMC-7721 cells and may have potential as an effective anti-cancer drug, acting to inhibit the Wnt/β-catenin signaling pathway.

Expression and purification of the recombinant murine REG3α protein in Pichia pastoris and characterization of its antimicrobial and antitumour efficacy

Regenerating islet-derived 3-alpha (REG3α) is a secreted intestinal antimicrobial protein, which shows antibacterial, anti-inflammatory and anti-apoptotic activities. It could significantly promote internal tissue regeneration and wound repair. In the present study, we expressed the codon-optimized murine REG3α in the yeast Pichia pastoris system. The secreted murine REG3α was captured using ProteinIsoTM Ni-NTA resin and further purified using a strong anion exchange resin Poros® 50 HQ. The final protein yield was 20 mg/L. The antibacterial activity and the anticancer efficacy of the recombinant REG3α were assessed using liquid growth inhibition assay, killing kinetics and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The results showed that the recombinant murine REG3α possessed antimicrobial activity against Escherichia coli, Salmonella paratyphi A, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus pumilus and Micrococcus luteus. Moreover, 100% killing against E. coli and S. aureus was observed after 30 min. The recombinant murine REG3α had a potent antitumour activity in a time-dependent and dose-dependent manner and had a negligible haemolysis activity against human erythrocytes. Taken together, P. pastoris is an efficient expression system for producing large quantities of antibacterial active REG3α for further research studies and clinical applications.