线虫surface coat proteins提取方法的建立与双向电泳分析

目的:建立一套适用于蛋白质双向电泳体系的线虫surface coat proteins(SCPs)样品制备技术,为今后研究线虫surfacecoat蛋白质组学及线虫病理生理学奠定基础。方法:以秀丽隐杆线虫(Caenorhabditis elegans)为研究材料,对比和分析不同的蛋白提取沉淀方法,进而采用SDS-PAGE电泳技术和双向电泳技术对所提蛋白进行评价。结果:通过35%乙醇结合TCA-丙酮沉淀法获得的质量较好的线虫SCPs,在12%的SDS-PAGE分析中该法提取的蛋白背景浅,蛋白条带多且清晰尖锐,含有丰富的蛋白信息量。通过双向电泳分析,可从提取的蛋白中鉴定出清晰蛋白点400多个。随机选择5个蛋白斑点,进行基质辅助激光解吸电离飞行时间质谱鉴定,鉴定得到高度匹配的已知线虫蛋白质2个。结论:所建立的方法可为今后研究线虫surface coat蛋白质组学及线虫病理生理学提供重要工具。

Cloning and Prokaryotic Expression of P23 Major Surface Protein Gene from Theileria sergenti

[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surface protein of T. sergenti (D84447).The P23 gene was amplified by PCR from genomic DNA of T. sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23. Positive clones were identified by PCR screening and restriction digestion. A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E. coli BL21. After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting. [Result] The cloned gene has a total length of 507 bp. Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank (D84447). The expressed fusion protein was 46 ku in molecular mass. Induction opportunity of zhours after culture inoculation was the best,the induction time of 6 h was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1 mmol/L had little effect on the expression. Western-blotting indicated that recombinant protein was recognized by specific antibody. [Conclusion] This study would lay a foundation for further research on the prevention and diagnose of T. sergenti.

Hepatocellular carcinoma and hepatitis B surface protein

The tumorigenesis of hepatitis B virus(HBV)-associated hepatocellular carcinoma(HCC) has been widely studied. HBV envelope proteins are important for the structure and life cycle of HBV, and these proteins are useful for judging the natural disease course and guiding treatment. Truncated and mutated pre S/S are produced by integrated viral sequences that are defective for replication. The pre S/S mutants are considered "precursor lesions" of HCC. Different pre S/S mutants induce various mechanisms of tumorigenesis, such as transactivation of transcription factors and an immune inflammatory response, thereby contributing to HCC. The pre S2 mutants and type Ⅱ "Ground Glass" hepatocytes represent novel biomarkers of HBVassociated HCC. The pre S mutants may induce the unfolded protein response and endoplasmic reticulum stress-dependent and stress-independent pathways. Treatments to inhibit hepatitis B surface antigen(HBs Ag) and damage secondary to HBs Ag or the pre S/S mutants include antivirals and antioxidants, such as silymarin, resveratrol, and glycyrrhizin acid. Methods for the prevention and treatment of HCC should be comprehensive.

Optimization of High-Protein Glutinous Rice Flour Production Using Response Surface Method

A response surface method was employed to study the effect of α-amylase concentration, hydrolysis temperature and time on the production of high protein glutinous rice flour(HPGRF). The suspension of glutinous rice flour(15%) that contained 6.52% protein was gelatinized and subsequently hydrolyzed by thermostable α-amylase. The hydrolysis yielded 0.144–0.222 g/g HPGRF with 29.4%–45.4% protein content. Hydrolysis time exerted a significant effect, while enzyme concentration and hydrolysis temperature showed insignificant effect on the protein content and production yield of HPGRF. The result of response surface method showed that the optimum condition for the production of HPGRF that contained at least 36% protein was treating gelatinized 15% glutinous rice flour suspension with 0.90 Kilo Novo α-amylase Unit(KNU)/g α-amylase at 80 oC for 99 min. By carrying out the predicted hydrolysis condition, HPGRF with 35.9% protein and 61.8% carbohydrates was resulted. The process yielded 0.172 g/g HPGRF. HPGRF contained higher amount of essential amino acids compared to glutinous rice flour. HPGRF had higher solubility and lower swelling power, and also showed no pasting peak compared with glutinous rice flour.

Comparative Study on the Infectivity and Spore Surface Protein of Nosema bombycis and Its Morphological Variant Strain

A new morphological variant strain of microsporidium was produced by infecting the mulberry looper, Hemerophilaatrilineata [Phthonandria atrilineata], with Nosema bombycis successively for 24 times, and named 24Nbh. Comparativestudies on morphology, infectivity and spore surface protein were conducted. 24Nbh was short and wide, and had asignificant difference (P<0.01) over the Nb spores. The infectivity tests conducted on second instar silkworm larvaeshowed that IC50 of 24Nbh was 1.98104 spores mL-1 and of Nb was 1.72103 spores mL-1, thus indicating that the infectivityof Nb decreased 11.5 times after multiplying in mulberry looper for 24 times. The IC50 of spores from silkworm infected with24 Nbh was 6.9 times less than Nb, showing that the infectivity of 24Nbh spores rejuvenated very fast when reinfected tosilkworms, further more, the length and width of such spore was larger than 24Nbh (P<0.01) and smaller than Nb (P<0.05).The SDS-PAGE profiles of Nb and 24Nbh were generally the same, 4 distinct proteins of 12, 17, 30, 33 kDa were obtainedwith difference in quantity. When 120 g of protein was applied for 2D-PAGE, five suspected different proteins withdifference in quantity were observed. These results demonstrate that these differential proteins maybe associated withvariation in infectivity of the spores.

The influence of phytase, pre-pellet cracked maize and dietary crude protein level on broiler performance via response surface methodology

Background:The reduction of crude protein levels in diets for broiler chickens may generate economic,environmental and flock welfare and health benefits;however,performance is usually compromised.Whole grain feeding and phytase may improve the utilization of reduced crude protein diets.Results:The effects of pre-pellet cracked maize(0,15%and 30%)and phytase(0,750 and 1500 FTU/kg)in isoenergetic maize-soy diets with three levels of crude protein(22%,19.5%and 17%)were evaluated via a BoxBehnken response surface design.Each of 13 dietary treatments were offered to 6 replicate cages(6 birds/cage)of male Ross 308 broiler chicks from 7 to 28 d post-hatch.Model prediction and response surface plots were generated from experimental data via polynomial regression in R and only significant coefficients were included and discussed in the predicted models.Weight gain,feed intake and FCR were all influenced by pre-pellet cracked maize,phytase and crude protein level,where crude protein level had the greatest influence.Consequently,the reduction from 22%to 17%dietary crude protein in non-supplemented diets reduced weight gain,feed intake,relative gizzard weight,relative gizzard content and relative pancreas weight but improved FCR.However,the inclusion of 30%cracked maize to 17%crude protein diets restored gizzard weight and 1500 FTU phytase inclusion to 17%crude protein diets increased relative gizzard contents and pancreas weights.Cracked maize and phytase inclusion in tandem to 17%crude protein diets increased weight gain,feed intake and FCR;however,this FCR was still more efficient than broilers offered the non-supplemented 22%crude protein diet.Broilers offered the prepellet cracked maize and phytase inclusions reduced AME in 22%crude protein diets but improved AME by 2.92 MJ(14.16 versus 11.24 MJ;P<0.001)in diets containing 17%crude protein.Ileal N digestibility was greater in broilers offered diets with 17%crude protein than those offered the 22%crude protein diet;irrespective of phytase and pre-pellet cracked maize.Conclusion:Pre-pellet cracked maize and phytase inclusions will improve the performance of broilers offered reduced crude protein diets.

Design and regulation of the surface and interfacial behavior of protein molecules

Surface and interfacial behavior of protein molecules are crucial for the protein function involved in many biochemical processes and biomedical products such as enzyme design,bio-separation,drug design and delivery.This article is devoted to an overview of design and regulation of the surface and interfacial behavior of protein molecules.The improvement of enzyme surface such as the directed evolution and the rational design of enzymes is introduced at first,followed by the rational design of protein interface for the protein assembly.Thereafter,the design of micro-environment and ligands are described as two examples for the design guided by protein surface.Then the design of protein surface and interface with the help of artificial intelligence will be discussed.

Novel Evidence Suggests Hepatitis B Virus Surface Proteins Participate in Regulation of HBV Genome Replication

Naturally occurring mutations in surface proteins of Hepatitis B virus(HBV) usually result in altered hepatitis B surface antigen(HBsAg) secretion efficiency.In the present study,we reported two conserved residues,M75 and M103 with respect to HBsAg,mutations of which not only attenuated HBsAg secretion(M75 only),but also suppressed HBV genome replication without compromising the overlapping p-gene product.We also found M75 and M103 can initiate truncated surface protein(TSPs) synthesis upon over-expression of full-length surface proteins,which may possibly contribute to HBV genome replication.However,attempts to rescue replicationdefective HBV mutant by co-expression of TSPs initiated from M75 or M103 were unsuccessful,which indicated surface proteins rather than the putative TSPs were involved in regulation of HBV genome replication.

Surface Display of Domain Ⅲ of Japanese Encephalitis Virus E Protein on Salmonella Typhimurium by Using an Ice Nucleation Protein

A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invade host cells, Salmonella typhimurium might be a good candidate for displaying viral antigens. We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S. typhimurium BRD509 using the ice nucleation protein. The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated. The results showed that display motifs in the protein can target integral foreign protein on the surface of S. typhimurium BRD509. Moreover, recombinant strains with surface displayed viral proteins retained their invasiveness, suggesting that the recombinant S. typhimurium can be used as live vaccine vector for eliciting complete immunogenicity. The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.

Immobilization of His-Tagged Proteins on Various Solid Surfaces Using NTA-Modified Chitosan

Continued advancement of protein array, bioelectrode, and biosensor technologies will necessitate development of methods that allow for increased protein immobilization capacity and more control over protein orientation. Toward these ends, we developed a method involving modification of chitosan with nitrilotriacetic acid (NTA) to achieve immobilization of a larger amount of His-tagged protein than is possible with current methods. The immobilization capacity of our method was evaluated using His-tagged GFP (Green Fluorescent Protein) as a model protein. The average immobilization density on modified glass was about 32 ng/mm2. Our method is suitable for use on a variety of solid surfaces, including glassy carbon, silicon wafers, polycarbonate, and beaten gold.

Sensitive and Label-Free Detection of Protein Secondary Structure by Amide Ⅲ Spectral Signals using Surface-Enhanced Raman Spectroscopy

Proteins and peptides perform a vital role in living systems, however it remains a challenge for accurate description of proteins at the molecular level. Despite that surface-enhanced Raman spectroscopy (SERS) can provide the intrinsic fingerprint information of samples with ultrahigh sensitivity, it suffers from the poor reproducibility and reliability. Herein, we demonstrate that the silver nanorod array fabricated by an oblique angle deposition method is a powerful substrate for SERS to probe the protein secondary structures without exogenous labels. With this method, the SERS signals of two typical proteins (lysozyme and cytochrome c) are successfully obtained. Additionally, by analyzing the spectral signals of the amide Ⅲ of protein backbone, the influence of concentration on the folding status of proteins has been elucidated. With the concentration increasing, the components of α-helix and β-sheet structures of lysozyme increase while the secondary structures of cytochrome c almost keep constant. The SERS method in this work offers an effective optical marker to characterize the structures of proteins.

SURFACE MODIFICATION OF TITANIUM FILMS WITH SODIUM ION IMPLANTATION:SURFACE PROPERTIES AND PROTEIN ADSORPTION

Sodium implanted titanium films with different ion doses were characterized to correlate their ion implantation parameters. Native titanium films and ion implanted titanium films were characterized with combined techniques of X-ray photoelectron spectroscopy （XPS）, atomic force microscopy （AFM）, and light microscopy （LM）. The surface presented increased sodium concentration on treated titanium films with ion dose increasing, except for the group with the highest ion dose of 4×10^17ions/cm^2. XPS depth profiling displayed that sodium entered titanium film around 25-50nm depth depending on its implantation ion dose. AFM characterization showed that sodium ion implantation treatment changed the surface morphology from a relatively smooth titanium film to rough surfaces corresponding to different implantation doses. After sodium implantation, implanted titanium films presented big particles with island structure morphology. The surface morphology and particle growth displayed the corresponding trend. Fibrinogen adsorption on these titanium films was performed to correlate with the surface properties of treated titanium films. The results show that protein adsorption on ion-implanted samples with dose of 2×10^17 and 4×10^17 are statistically higher （p 〈0.01） than samples treated with dose of 5×10^16 and 1×10^17, as well as the control samples.

A novel detection of single-stranded DNA binding protein based on ss-DNA modified chip using surface plasmon resonance microscopy

An ss-DNA gold chip was prepared based on self-assembly of the thiol-derivatized oligonucleotide, and used for the determination of single-stranded binding protein （SSB） by surface plasmon resonance microscopy （SPR）. The experiment results showed that SSB binds ss-DNA with high specificity, and relative signal of SPR response is proportional to the concentration of SSB in the range of 0.1-100 ng/mL with a detection limit （S/N = 3） of 0.07 ng/mL.

Construction and in vitro Expression of Streptococcus Mutans Surface Protein Encoding DNA Vaccine

DNA vaccine plasmids were constructed that encoded two highly conservative regions of a surface protein, PAc, from the human major cariogenic bacterium, Streptococcus mutans . Antigen expression was evaluated in vitro by immunohistochemical analysis of human endothelial cells following cationic liposome mediated transient transfection with recombinant plasmid. The results of this study provided a basis for further testing of these recombinant plasmids in primates and for efficacy testing of dental caries DNA vaccines in human volunteers in future.

Protein surface recognition of the novel tetra-carboxylphenyl calix[4]arene to cytochrome c

The interaction of the novel tetra-carboxylphenyl calix[4]arene （TCPC） with the bovine heart cytochrome c （Cc） was first investigated by fluorescence spectroscopy and molecular modeling methods. The formation of a stable 1：1 complex was monitored by fluorescence titration, and its binding constant is 1.916 ×10^7 L mol^-1. Molecular modeling reveals the recognition mechanism of TCPC to the Cc surface, that is, the electrostatic interaction drives TCPC to the Cc surface, and the van der Waals interaction orientates TCPC parallel to the cleft of Cc.

The involvement of proline-rich protein Mus musculus predicted gene 4736 in ocular surface functions

AIM: To research the two homologous predicted proline -rich protein genes, Mus musculus predicted gene 4736 (MP4) and proline-rich protein BstNI subfamily 1 (Prb1) which were significantly upregulated in cultured corneal organs when encountering fungal pathogen preparations. This study was to confirm the expression and potential functions of these two genes in ocular surface. METHODS: A Pseudomonas aeruginosa keratitis model was established in Balb/c mice. One day post infection, mRNA level of MP4 was measured using real-time polymerase chain reaction (PCR), and MP4 protein detected by immunohistochemistry (IHC) or Western blot using a customized polyclonal anti -MP4 antibody preparation. Lacrimal glands from normal mice were also subjected to IHC staining for MP4. An online bioinformatics program, BioGPS, was utilized to screen public data to determine other potential locations of MP4. RESULTS: One day after keratitis induction, MP4 was upregulated in the corneas at both mRNA level as measured using real -time PCR and protein levels as measured using Western blot and IHC. BioGPS analysis of public data suggested that the MP4 gene was most abundantly expressed in the lacrimal glands, and IHC revealed that normal murine lacrimal glands were positive for MP4 staining. CONCLUSION: MP4 and Prb1 are closely related with the physiology and pathological processes of the ocular surface. Considering the significance of ocular surface abnormalities like dry eye, we propose that MP4 and Prb1 contribute to homeostasis of ocular surface, and deserve more extensive functional and disease correlation studies.

Excess adsorption of biomolecules on soft surfaces: Adsorption of DNA, proteins and lactose on fatty surfaces

Insoluble fatty surfaces are involved in many important interactions such as in biomembranes with soluble biological macro and micromolecules. In this paper we have studied the adsorption interaction of aqueous solution of DNA, some proteins and lactose on several sparingly soluble fatty substances namely milk fat, stearic acid, palmitic acid, phosphatidyl choline and cholesterol surfaces by measuring the depletion of the adsorbates by analytical methods. Adsorption () of DNA on the soft surfaces of stearic acid, milk fat, phosphatidyl choline, palmitic acid and cholesterol was measured as a function of DNA concentration C2. In each case was found to increase with C2 until it reached the maximum value at a critical concentration . For different surfaces stands in the order: stearic acid > milk fat > phosphatidyl choline > cholesterol > palmitic acid. DNA forms multilayers on stearic acid surface. Adsorption of hemoglobin on cholesterol surface is found to be negative or zero but that of BSA on cholesterol is positive. Adsorption of gelatin on cholesterol surface is significantly higher than that of BSA. Lysozyme on cholesterol surface forms multilayers and on casein forms bilayer. The lowering of free energies ?DGo for all systems have been calculated using integrated form of the Gibbs adsorption and their values have been compared with each other. It is concluded that despite differences in the adsorption behavior of the biomolecules on various soft surfaces, free energy change expressed as Bull’s free energy change (Δ) remain nearly constant except for BSA-fatty acid interaction which may be likely due a specific interaction.

Surface Plasmon Resonance for C-Reactive Protein Detection in Human Plasma

In this work, we describe an approach of detecting biomarkers by Surface Plasmon Resonance imaging (SPRi) technique in real samples. Two C-Reactive Protein (CRP)-antibody immobilization methods were used: The first method was based on direct physisorption of CRP-antibody onto gold surface;the second one was based on oriented CRP-antibody with protein G intermediate layer. The two developed immunosensors were tested against CRP antigen in phosphate buffer saline solution with the SPRi technique. The response of the developed immunosensors was reproducible and stable. The detection limit of 10 pg&#183mL&#451 and 50 pg&#183mL&#451 CRP-antigen was observed with and without protein G respectively with this technique. Moreover, the developed SPRi immunosensor was used for CRP-antigen detection in human plasma. A detection limit of 5 ng&#183mL&#451 and 10 ng&#183mL&#451 was obtained with and without protein G respectively. These obtained results were compared to those obtained with QCM (Quartz Crystal Microbalance) and Enzyme-Linked Immunosorbent Assay (ELISA) techniques.

Cell Surface Display of Red-Grouper Nervous Necrosis Virus Capsid Protein on <i>Pichia pastoris</i>

Nervous necrosis virus (NNV), the etiological agent of viral nervous necrosis, has a high mortality rate of 100% in hatchery-reared larvae and juveniles. At present, there are still no effective vaccines available for NNV. Pichia pastoris surface display of viral capsid proteins was generated in hopes of developing an oral vaccine against red-grouper-nervous-necrosis virus (RGNNV) in fish. Fingerlings or juveniles that showed clinical signs of NNV infection were proved by RT-PCR for the appearance of expected length of 198 bpcDNA and further analysis by DNA sequencing. The DNA fragment containing AGα1 linked to RG-NNVRNA2, 2100 bp in length, was inserted into pPIC9K vector. Linearlized plasmids were electroporated into P. pastoris GS115 (mut+His?) and yeast isolates that had Muts?His+ and resistance phenotype at 4 mg/mL geniticin were selected to determine the integration of the target gene by PCR reaction. The extracted cell walls from the yeasts cultured in buffered-methanol-complex medium (BMMY) through an induction of 0.5% methanol for 6 days, were investigated for the fusion proteins by western blot. A protein band of 73 kDa predicted to be the fusion protein and a non-specific one of 56 kDa were detected. Staining of the fusion proteins expressing cells with corresponding antibodies revealed their presence of NNVRNA2, but varied the intensity of detected signals from cell to cell by confocal laser scanning fluorescence microscopy. The predicted fusion proteins tertiary structure also confirmed exposed conformation of the fusion protein on the cell wall. In this study, the capsid proteins from the red-spotted grouper nervous necrosis virus were successfully expressed on the cell surface of P. pastoris but still low levels of fusion protein expression. Further studies are required to optimize fully surface protein expression prior to evaluate the possible use of the constructed recombinant yeast as an oral vaccine against RG-NNV infection.

Optimization of Multigrain Premix for High Protein and Dietary Fibre Biscuits Using Response Surface Methodology (RSM)

In order to improve the nutritional quality of biscuits, a multigrain premix (MGP) was developed by using whole barley, sorghum, chickpea, pea and defatted soya flour, each at 20% level. The developed MGP had 26.28% protein, 10.13% insoluble dietary fiber and 7.38% soluble dietary fiber. The experiment was designed to optimise the MGP and wheat flour concentration for the development of multigrain biscuits with high protein, dietary fibre and to maximize the acceptability by the application of central composite rotatable design (CCRD) of Response Surface Methodology (RSM). The levels of incorporation of MGP and wheat flour were taken as variables whereas protein, soluble, insoluble fibers, biscuit dough hardness, breaking strength and overall acceptability (OAA) as responses. The optimum level of MGP and wheat flour obtained using numerical optimization was found to be 40 g and 60 g respectively. The biscuits prepared using these had 16.61% protein, 2.57% soluble fibre, and 6.67% insoluble fibre which is significantly (p ≤ 0.05) higher than control biscuit.