C-di-GMP Regulates Motile to Sessile Transition by Modulating MshA Pili Biogenesis and Near-Surface Motility Behavior in Vibrio cholerae.

In many bacteria, including Vibrio cholerae, cyclic dimeric guanosine monophosphate (c-di-GMP) controls the motile to biofilm life style switch. Yet, little is known about how this occurs. In this study, we report that changes in c-di-GMP concentration impact the biosynthesis of the MshA pili, resulting in altered motility and biofilm phenotypes in V. cholerae. Previously, we reported that cdgJ encodes a c-di-GMP phosphodiesterase and a ΔcdgJ mutant has reduced motility and enhanced biofilm formation. Here we show that loss of the genes required for the mannose-sensitive hemagglutinin (MshA) pilus biogenesis restores motility in the ΔcdgJ mutant. Mutations of the predicted ATPase proteins mshE or pilT, responsible for polymerizing and depolymerizing MshA pili, impair near surface motility behavior and initial surface attachment dynamics. A ΔcdgJ mutant has enhanced surface attachment, while the ΔcdgJmshA mutant phenocopies the high motility and low attachment phenotypes observed in a ΔmshA strain. Elevated concentrations of c-di-GMP enhance surface MshA pilus production. MshE, but not PilT binds c-di-GMP directly, establishing a mechanism for c-di-GMP signaling input in MshA pilus production. Collectively, our results suggest that the dynamic nature of the MshA pilus established by the assembly and disassembly of pilin subunits is essential for transition from the motile to sessile lifestyle and that c-di-GMP affects MshA pilus assembly and function through direct interactions with the MshE ATPase.

Overproduction of recombinant human mannose-binding lectin (MBL) in Chinese hamster ovary cells.

Mannose-binding lectin (MBL) is an important serum protein that functions in the innate immune system and has been considered to have therapeutic potential in MBL replacement therapies for patients with deficient or low levels of MBL. In this study, we established a Chinese hamster ovary (CHO) cell line that overexpresses the recombinant human MBL (rhMBL) protein. In an 11-day batch culture process using a 30-L bioreactor (20-L working volume) and serum-free medium, these cells could produce over 226 mg/L of rhMBL protein. The recombinant protein was then purified to homogeneity from the culture supernatant using a three-step chromatographic procedure that resulted in a recovery rate of approximately 55%. This purified rhMBL protein adopted oligomeric bouquet-like structures that were similar to those of native MBL present in human blood, and these oligomeric structures were reported to be critical in MBL functions. We further demonstrated in carbohydrate binding and complementation activation assays that this rhMBL protein was functionally active with very similar dissociation constants and half maximal effective concentrations to those of native MBL.

Expression analysis of a type S2 EUL-related lectin from rice in Pichia pastoris.

Rice (Oryza sativa) expresses different putative carbohydrate-binding proteins belonging to the class of lectins containing an Euonymus lectin (EUL)-related domain, one of them being OrysaEULS2. The OrysaEULS2 sequence consists of a 56 amino acid N-terminal domain followed by the EUL sequence. In this paper the original sequence of the EUL domain of OrysaEULS2 and some mutant forms have been expressed in Pichia pastoris. Subsequently, the recombinant proteins were purified and their carbohydrate binding properties determined. Analysis of the original protein on the glycan array revealed interaction with mannose containing structures and to a lesser extent with glycans containing lactosamine related structures. It was shown that mutation of tryptophan residue 134 into leucine resulted in an almost complete loss of carbohydrate binding activity of OrysaEULS2. Our results show that the EUL domain in OrysaEULS2 interacts with glycan structures, and hence can be considered as a lectin. However, the binding of the protein with the array is much weaker than that of other EUL-related lectins. Furthermore, our results indicate that gene divergence within the family of EUL-related lectins lead to changes in carbohydrate binding specificity.

[Mannose-binding lectin (MBL) inhibits human cytomegalovirus infection of human MD-DC].

OBJECTIVE: To explore the inhibitory effect of different sources, different concentrations of Mannose-binding lectin (MBL) on human cytomegalovirus infection of human MD-DC cells. METHODS: The recombinant MBL was acquired by vector construction, and the natural MBL was purified from human plamsa. MD-DC were pre-exposed to several dilutions of the hMBL/rMBL for 30 min, then HCMV suspensions were added to MD-DC for 2 h to compare the inhibitory effect of hMBL/rMBL on the HCMV infection of MD-DC. MD-DC infected by HCMV co-culture with hMBL/rMBL to compare the inhibitory effect of hMBL/rMBL on the HCMV diffusion between MD-DC. HCMV-DNA in MD-DC was detected by fluorescence quantitative PCR. HCMV-PP65 in MD-DC was analyzed with flow cytometry, the ability of MD-DC to capture HCMV was observed with immunofluorescence confocal microscope. RESULTS: In hMBL/rMBL inhibition the ability of MD-DC capture HCMV experiments, the fluorescent quantitative PCR demonstrated that the amount of HCMV-DNA in 1 microg/mL of hMBL/rMBL treated cells was not significantly different from that of control group (P < 0.05). But the HCMV-DNA in 5 microg/mL and 10 microg/mL hMBL/rMBL treated group were significantly lower than that of control group (P < 0.05). The significant inhibit effects of 10 microg/mL hMBL/rMBL on the ability of MD-DC capture HCMV were observed by immunofluorescence confocal microscopy and flow cytometry. The inhibit effects of hMBL/rMBL on HCMV diffusion between MD-DC were also observed in 5 microg/mL and 10 microg/mL hMBL/rMBL treated groups at 72 hours. CONCLUSION: The hMBL/rMBL in physiological concentration range (5-10 microg/mL) can significantly inhibit human cytomegalovirus infection of human MD-DC cells, and the hMBL is more effective than rMBL.

Evolutionary conservation of mannan-binding lectin (MBL) in bony fish: identification, characterization and expression analysis of three bona fide collectin homologues of MBL in the rainbow trout (Onchorhynchus mykiss).

The complement system of fish is generally as complex as in mammals, and in addition Teleost fish often possess several genes encoding different subtypes of a given complement component, such as C3-1, C3-3 and C3-4. Initiators of both the classical (C1) and alternative pathway (factor B) have been characterized in the rainbow trout but so far no molecules of the lectin pathway have been identified. Based on the generally accepted idea of complement evolution, which predicts that the alternative pathway predates the two other pathways, and that the lectin pathway developed before the classical, we set out to characterize members of the lectin pathway in fish. We identified and characterized three homologues of mannan-binding lectin (MBL) with a bona fide collectin structure. By means of RT-PCR and immunohistochemistry using monoclonal antibodies we found that they were synthesized in the spleen, the anterior intestine and the liver. In the liver, we saw co-expression with mannan-binding lectin associated serine protease (MASP). The MBL homologues 2 and 3 (MBL-H2,3) were also found in the vascular system of the rainbow trout. By means of gel size exclusion chromatography of serum we found that MBL-H2,3 oligomerized heterogeneously from monomers to tetramers of a trimeric collagenous subunit. Sequence comparison and phylogenetic studies showed that the homologues were more related with MBL than any other collectins, and that two previously characterized trout proteins, designated MBL1 and MBL2, should be reconsidered as MBL candidates.

Dendrobium findleyanum agglutinin: production, localization, anti-fungal activity and gene characterization.

The recently reported Dendrobium findleyanum agglutinin (DFA) was identified and determined in different parts of D. findleyanum pseudobulbs by using Western blot analysis, LC-MS/MS, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and histochemical procedure. Western blot analysis of crude protein extract with horseradish peroxidase (HRP), a mannose-rich glycoprotein, showed only one band at 14.5 kDa, which had the same molecular mass as DFA. This band was a major band when the membrane was stained with Coomassie Brilliant Blue. The protein profiles from SDS-PAGE showed higher band intensity of the 14.5 kDa mannose-binding protein in nearly mature and mature stages, compared to very young and young stages of the orchid. In addition, the band intensity was to a great extent different between the swollen and the non-swollen internode of the pseudobulb. Using LC-MS/MS, the sequence tags of the 14.5-kDa protein bands from the node, swollen internode and non-swollen internode revealed that the protein was DFA. Histochemical procedure in the transverse section of the pseudobulbs demonstrated major HRP binding sites, which reflected the location of DFA, in periphery of parenchymal cells. The purified DFA showed anti-fungal activity against Alternaria alternata and Collectotrichum sp. Using reverse transcription polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of the DFA precursor revealed 94% homology with a lectin precursor from D. officinale. N-terminal sequencing demonstrated the processing site between residues 24 and 25 of the DFA precursor.

Gene polymorphisms associated with reduced hepatic expression of porcine mannan-binding lectin C.

Previous studies showed that low expression of mannan-binding lectin C (MBL-C) in pigs was not due to single-nucleotide polymorphisms (SNPs) in the coding region of pig MBL2. In these studies, we compared the 5' flanking regions of porcine MBL1 (1907 bp) and MBL2 (1880 bp) in normal and diseased pigs with low or high hepatic expression of MBL2. Hepatic expression of MBL-C was very low in all pigs submitted for postmortem diagnosis. In various European pig breeds, a G(-1081)A substitution was linked to very low hepatic MBL-C expression, and was more frequent in diseased pigs. A C(-251)T substitution with less influence on MBL-C expression was more common in various breeds but was not associated with disease. MBL2 polymorphisms were associated with some disease groups and with the presence of some etiologic agents. These findings indicate that some promoter polymorphisms impair MBL-C expression in pigs and may increase their susceptibility to disease.

The expression of innate immunity genes in Italian Crohn disease patients.

Crohn' disease (CD) is a chronic idiopathic inflammatory bowel disease characterized by the interaction of both hereditary and environmental factors. Intestinal flora and pathogens such as bacteria, viruses and fungi, are thought to be the first step leading to an inflammatory status, which is subsequently amplified in genetically susceptible patients thus triggering the disease. Since the innate immune system is believed to be very important in regulating the flora of the gastrointestinal tract, we decided to study the influence of two important molecules of the innate immune system in CD. Frozen intestinal biopsies from 49 Crohn patients and 10 healthy individuals were collected at the gastroenterology unit of Children's Hospital Burlo Garofolo in Trieste and innate immunity gene expression was evaluated by using both in situ RT-PCR and quantitative PCR. We have analyzed the expression and localization of both MBL2 and DEFB1 genes in intestinal biopsies of Italian Crohn patients by in situ RT-PCR and quantitative PCR. DEFB1 is expressed equally in all subjects. Importantly, MBL2 transcripts were upregulated in CD patients compared to healthy controls. MBL2 expression in controls is normally extremely low, detectable only by quantitative PCR with a Taqman probe. We demonstrated the MBL2 and DEFB1 expression in intestinal biopsies of patients suffering from CD. Our results showed that the MBL2 gene is expressed by cells in the basal lamina, whilst DEFB1 is expressed by epithelial cells.

Mannan-Binding Lectin Is Involved in the Protection against Renal Ischemia/Reperfusion Injury by Dietary Restriction.

Preoperative fasting and dietary restriction offer robust protection against renal ischemia/reperfusion injury (I/RI) in mice. We recently showed that Mannan-binding lectin (MBL), the initiator of the lectin pathway of complement activation, plays a pivotal role in renal I/RI. Based on these findings, we investigated the effect of short-term DR (30% reduction of total food intake) or three days of water only fasting on MBL in 10-12 weeks old male C57/Bl6 mice. Both dietary regimens significantly reduce the circulating levels of MBL as well as its mRNA expression in liver, the sole production site of MBL. Reconstitution of MBL abolished the protection afforded by dietary restriction, whereas in the fasting group the protection persisted. These data show that modulation of MBL is involved in the protection against renal I/RI induced by dietary restriction, and suggest that the mechanisms of protection induced by dietary restriction and fasting may be different.

The site-directed mutagenesis of gastrodia anti-fungal protein mannose-binding sites and its expression in Escherichia coli.

Gastrodia anti-fungal protein (GAFP) displays strong inhibitory activity against certain fungal pathogens. Five GAFP analogues with different mutations at mannose-binding sites and the wild-type one were expressed and purified in Escherichia coli. The inhibitory analysis of the purified various GAFPs against the growth of Trichoderma viride indicates that single amino acid mutated-type GAFPs have inhibitory activity, but its activity is much less than the wild-type one. The double and triplicate amino acids mutated GAFPs have very low inhibitory activity. For the first time it was proved that GAFP mannose-binding sites play key role in anti-fungi process.

[Construction of the expression vector PcDNA4/His C-MBL and its expression in Chinese-hamster ovary cells].

OBJECTIVE: To construct pcDNA4/His C-MBL recombinant eukaryotic expression plasmid and examine its expression of mannan-binding lectin (MBL) in mammary cells. METHODS: The target sequence was amplified by PCR from pGEM-MBL plasmid that contains wild-type human MBL cDNA, and inserted into eukaryotic expression vector PcDNA4/His C followed by restriction mapping and sequencing. The recombinant plasmid PcDNA4/His C-MBL was transformed into Chinese-hamster ovary (CHO) cells by electroporation, and the Zeocin-resistant clones were selected for analysis of mRNA expression by reverse transcription (RT)-PCR. The expressed product was purified by immobilized metal affinity chromatography (IMAC) and identified by SDS-PAGE and Western-blot analysis, and its immunoreactivity was detected by an indirect enzyme-linked immunosorbent assay ELISA using the anti-serum from Balb/C mice immunized with the recombinant protein. RESULTS: The cDNA fragment of 750 bp was amplified from pGEM-MBL plasmid, which was shown by restriction enzyme digestion and DNA sequencing. The mRNA expression of Zeocin-resistant CHO cell clones was detected by RT-PCR. Three components of 29, 58 and 87 kD in the purified recombinant product were found by SDS-PAGE and the 29 kD component could be recognized by anti-6His antibody in Western blot analysis. The titers of the anti-serum from immunized mice were 1: 819 200 against the recombinant protein and 1:25 600 against both the natural human MBL and the recombinant trimeric carbohydrate-recognition domain (CRD) of human MBL, as determined by the indirect ELISA. CONCLUSION: The cell strains that express recombinant human MBL (rhMBL) and rhMBL protein have been obtained successfully, which provides the basis for further research of MBL molecule.

Functional characterization of mannose-binding lectin in zebrafish: implication for a lectin-dependent complement system in early embryos.

The lectin pathway involves recognition of pathogen-associated molecular patterns by mannose-binding lectin (MBL), and the subsequent activation of associated enzymes, termed MBL-associated serine proteases (MASPs). In this study, we demonstrate that the transcript of MBL gene is present in the early embryo of zebrafish, and MBL protein is also present in the embryo. In addition, we show that recombinant zebrafish MBL was able to bind the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, and rMBL was able to promote the phagocytosis of E. coli and S. aureus by macrophages, indicating that like mammalian MBL, zebrafish MBL performs a dual function in both pattern recognition and opsonization. Importantly, we show that microinjection of anti-MBL antibody into the early developing embryos resulted in a significantly increased mortality in the embryos challenged with Aeromonas hydrophila (pathogenic to zebrafish); and injection of rMBL into the embryos (resulting in increase in MBL in the embryo) markedly promoted their resistance to A. hydrophila; and this promoted bacterial resistance was significantly reduced by the co-injection of anti-MBL antibody with rMBL but not by the injection of anti-actin antibody with rMBL. These suggest that the lectin pathway may be already functional in the early embryos in zebrafish before their immune system is fully matured, protecting the developing embryos from microbial infection. This work provides a new angle to understand the immune role of the lectin pathway in early development of animals.

Adjuvant effects of mannose-binding lectin ligands on the immune response to infectious bronchitis vaccine in chickens with high or low serum mannose-binding lectin concentrations.

Mannose-binding lectin (MBL) plays a major role in the immune response as a soluble pattern-recognition receptor. MBL deficiency and susceptibility to different types of infections have been subject to extensive studies over the last decades. In humans and chickens, several studies have shown that MBL participates in the protection of hosts against virus infections. Infectious bronchitis (IB) is a highly contagious disease of economic importance in the poultry industry caused by the coronavirus infectious bronchitis virus (IBV). MBL has earlier been described to play a potential role in the pathogenesis of IBV infection and the production of IBV-specific antibodies, which may be exploited in optimising IBV vaccine strategies. The present study shows that MBL has the capability to bind to IBV in vitro. Chickens from two inbred lines (L10H and L10L) selected for high or low MBL serum concentrations, respectively, were vaccinated against IBV with or without the addition of the MBL ligands mannan, chitosan and fructooligosaccharide (FOS). The addition of MBL ligands to the IBV vaccine, especially FOS, enhanced the production of IBV-specific IgG antibody production in L10H chickens, but not L10L chickens after the second vaccination. The addition of FOS to the vaccine also increased the number of circulating CD4+ cells in L10H chickens compared to L10L chickens. The L10H chickens as well as the L10L chickens also showed an increased number of CD4-CD8α-γδ T-cells when an MBL ligand was added to the vaccine, most pronouncedly after the first vaccination. As MBL ligands co-administered with IBV vaccine induced differences between the two chicken lines, these results indirectly suggest that MBL is involved in the immune response to IBV vaccination. Furthermore, the higher antibody response in L10H chickens receiving vaccine and FOS makes FOS a potential adjuvant candidate in an IBV vaccine.

Complement polymorphisms and cognitive dysfunction after carotid endarterectomy.

OBJECT: The role of genetic polymorphisms in the neurological outcome of patients after carotid endarterectomy (CEA) remains unclear. There are single nucleotide polymorphisms (SNPs) that predispose patients to postoperative cognitive dysfunction (CD). We aim to assess the predictability of three complement cascade-related SNPs for CD in patients having CEAs. METHODS: In 252 patients undergoing CEA, genotyping was performed for the following polymorphisms: complement component 5 (C5) rs17611, mannose-binding lectin 2 (MBL2) rs7096206, and complement factor H (CFH) rs1061170. Differences among genotypes were analyzed via the chi-square test. Patients were evaluated with a neuropsychometric battery for CD 1 day and 1 month after CEA. A multiple logistic regression model was created. All variables with univariate p < 0.20 were included in the final model. RESULTS: The C5 genotypes A/G (OR 0.26, 95% CI 0.11-0.60, p = 0.002) and G/G (OR 0.22, 95% CI 0.09-0.52, p < 0.001) were significantly associated with lower odds of exhibiting CD at 1 day after CEA compared with A/A. The CFH genotypes C/T (OR 3.37, 95% CI 1.69-6.92, p < 0.001) and C/C (OR 3.67, 95% CI 1.30-10.06, p = 0.012) were significantly associated with higher odds of exhibiting CD at 1 day after CEA compared with T/T. Statin use was also significantly associated with lower odds of exhibiting CD at 1 day after CEA (OR 0.43, 95% CI 0.22-0.84, p = 0.01). No SNPs were significantly associated with CD at 1 month after CEA. CONCLUSIONS: The presence of a deleterious allele in the C5 and CFH SNPs may predispose patients to exhibit CD after CEA. This finding supports previous data demonstrating that the complement cascade system may play an important role in the development of CD. These findings warrant further investigation.

[Preparation of the trimeric subunits of recombinant human mannan-binding lectin and analysis of its bioactivity].

OBJECTIVE: To prepare the trimeric subunits of recombinant human mannan-binding lectin (MBL) with biological activities. METHODS: A prokaryotic expression vector containing human MBL N-terminal deletant (rhMBLδN) gene we previously constructed was transformed into E. coli for efficient expression of rhMBLδN fusion protein. Based on the principle that the collagen polypeptides tend to self-assembly into the tertiary structure of proteins by forming a triple helix due to the characteristic properties of the collagen proteins, rhMBLδN fusion protein was limitedly hydrolyzed with thrombin. The obtained rhMBLδN polypeptide was repeatedly dialyzed in 50 mmol/L PBS (pH7.2) and ddH(2)O, and the final product was analyzed for its bioactivities using a ligand-binding assay and a C4d deposition assay. RESULTS: rhMBLδN polypeptide with a relative molecular mass of about 20 000 was obtained by limited proteolysis of rhMBLδN fusion protein with thrombin. Repeated dialyses of rhMBLδN polypeptides in 50 mmol/L PBS and ddH(2)O resulted in the isolation of the trimeric subunit trhMBLδN (with a relative molecular mass of about 50 000), which contained a collagen-like helix. The trhMBLδN protein had a higher ligand-binding activity than rhMBLδN polypeptide, and acquired the activity to initiate the lectin pathway of complement activation, but the activities were lower than those of natural MBL. CONCLUSION: We have successfully obtained the bioactive trimeric subunit of rhMBL, trhMBLδN, and this structural subunit is also the functional subunit of the MBL molecule.

High polymorphism of the MBL2 gene in patients with atopic dermatitis.

BACKGROUND: Low serum levels of mannose-binding lectin (MBL) are determined mainly by variant alleles of the MBL2 gene and it has been suggested that MBL may play a role in the susceptibility to atopic dermatitis (AD). OBJECTIVE: The aim was to investigate the difference of the frequency of MBL2 variant alleles in AD patients and in a group of individuals without AD, and associate the MBL2 alleles with AD severity. METHODS: MBL2 variant allele's frequency was investigated in 131 children with AD and 165 healthy children/adolescents matched by convenience. The severity of disease was graded according to the SCORing Atopic Dermatitis (SCORAD) index. The first exon variants were called "O" and the wild type "A". The variants in the promoter were H/L at -550 and X/Y at -221, determined by Real Time PCR. RESULTS: Children with AD had higher frequency of allele O and the genotypes related to low or deficient levels of MBL, when compared to the healthy group (p = 0.0012 and p < 0.001, respectively), but not with AD severity. CONCLUSION: Low or deficient MBL serum levels determined genetically may contribute to the predisposition for AD, but not for disease severity.

Spatiotemporal activity of the mshA gene system in Shewanella oneidensis MR-1 biofilms.

Type IV pili and a putative EPS biosynthetic gene cluster (mxdABCD) have been implicated previously in biofilm formation in Shewanella oneidensis MR-1. Here, we report that the mannose-sensitive hemagglutinin (MSHA) pilus mediates a reversible, d-mannose-sensitive association of cells to the substratum surface or to other cells that is critical within the first 5 microm of the biofilm from the substratum. The presence of the MSHA pilus alone is insufficient to confer biofilm-forming capacity; its activity, as mediated by the putative pilus retraction motor protein, PilT, is also required. Deletion of pilD, encoding the type IV pili prepilin peptidase, revealed that additional PilD substrate(s) may be involved in biofilm formation beyond the major structural pilin of the MSHA pilus. We also present data showing that the MSHA pilus and mxd genes encode for a complementary set of molecular machineries that constitute the dominant mechanisms enabling biofilm formation in this microorganism under hydrodynamic conditions.

Mannose-binding lectin is produced by vaginal epithelial cells and its level in the vaginal fluid is influenced by progesterone.

Mannose-binding lectin (MBL) is a recognition molecule of the complement (C) system and binds to carbohydrate ligands present on a wide range of pathogenic bacteria, viruses, fungi, and parasites. MBL has been detected in the cervico-vaginal cavity where it can provide a first-line defence against infectious agents colonizing the lower tract of the reproductive system. Analysis of the cervico-vaginal lavage (CVL) obtained from 11 normal cycling women at different phases of the menstrual cycle revealed increased levels of MBL in the secretive phase. Part of this MBL derives from the circulation as indicated by the presence of transferrin in CVL tested as a marker of vascular and tissue permeability. The local synthesis of MBL is suggested by the finding that its level is substantially higher than that of transferrin in the secretive phase. The contribution of endometrium is negligible since the MBL level did not change before and after hysterectomy. RT-PCR and in situ RT-PCR analysis showed that the vaginal tissue, and in particular the basal layer of the epithelium, is a source of MBL which binds to the basal membrane and to cells of the outer layers of the epithelium. In conclusion, we have shown that MBL detected in CVL derives both from plasma as result of transudation and from local synthesis and its level is progesterone dependent increasing in the secretive phase of the menstrual cycle.

[Prokaryotic expression of Balb/C mouse MBL-A carbohydrate recognition domain].

OBJECTIVE: To express the carbohydrate recognition domain (CRD) of Balb/C mouse mannan binding lectin A (MBL-A) in E.coli. METHODS: The target gene fragment was obtained by PCR from the plasmid pmMBL-A harboring mouse MBL-A gene. The PCR product was recombined with the prokaryotic expression vector pET-41a(+) and the resulting recombinant plasmid was identified by PCR, restriction analysis and sequencing before transformation into E.coli BL21(DE3) cell for expression of the target protein. After washing and renaturation, the protein was purified on GST-Tag purification resins and analyzed by SDS-PAGE, Western blotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: A DNA fragment of about 450 bp was amplified by PCR and the recombinant plasmid pET41a-mMBL-A-CRD was constructed by linking the fragment with pET41a(+) vector. The result of restriction enzyme analysis and sequencing of the selected clones were consistent with those by computer analysis. The recombinant vector was expressed in E.coli BL21(DE3), and the expressed protein existed mainly as inclusion bodies, whose relative molecular mass was about 47,000 by SDS-PAGE analysis. After washing, renaturation and purification, the purity of recombinant protein was about 90%. Western blotting suggested immunoreactivity of the purified protein with anti-GST antibody, and its sugar binding activity was verified by ELISA. CONCLUSION: We have successfully obtained mouse MBL-A CRD protein, which provides the base for further functional study of the MBL-A molecule.

[Cloning, sequencing analysis and expression of a putative mannose-binding lectin gene from Polygonatum roseum in Xinjiang].

The genomic DNA were extracted from the leaves of Polygonatum roseum (Liliaceae) in Xinjiang and the primers were designed according to conservative sequences of Polygonatum lectins gene. The complete ORF of Polygonatum roseum agglutinin (PRA) gene was amplified as a fragment of 550 bp, which was identical with predicted size. Like most of the plant lectin genes, there was no intron in the PRA gene. The ORF of the gene encoded 159 amino acid residues, in which included a signal sequence of 28 amino acid residues at its N-terminus. The cDNA sequence had 92% identities compared with the published sequence. The amino acid sequence and SWISS-MODEL analysis indicated that the three-dimensional structure of PRA strongly resembled with that of monocot mannose-binding lectins, which comprised with three antiparallel four-stranded beta-sheets arranged as a 12-stranded beta-barrel. The recombinant pGEX4T-1-PRA and pMAL-p2x-PRA prokaryotic expression vectors were constructed to produce GST-PRA and MBP-PRA fusion proteins in E. coli, respectively. SDS-PAGE of the fusion protein demonstrated that the PRA lectin protein migrated at a size of 14 kD. The immunization was performed by intra-muscular injection of pcDNA3-PRA, and the antiserum was detected by ELISA. Western blotting analysis showed the antiserum specifically bound the lectin protein. The establishment of such an expression system might provide materials for further investigation of the properties and functions of PRA proteins. It also laid the basis for plant genetic engineering on its defensive functions to pests and diseases.