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.

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.

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.

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.

Protein Based Localized Surface Plasmon Resonance Gas Sensing

We apply the localized surface plasrnon resonance （LSPR） of gold nanoparticles （GNPs） covalently coupled with cytochrorne c （cyt c） to create a nanobiosensor for detecting hydrogen sulfide （H2S） in the range of 15 lOOppb. Monolayer formation of GNPs on glass surface functionalized with 3-aminopropyltrirnethoxysilane （APTMS） is performed for fabricating a chip-based format of the optical transducer. By chemical introduction of short-chain thiol derivatives on cyt c protein shell via its lysine residues, a very fast self-assembled rnonolayer （SAM） of cyt c is formed on the GNPs. Significant shifts in the LSPR peak （△λLSPR） are observed by reacting H2S with cyt c. Results show a linear relationship between △λLSPR and H2S concentration. Furthermore, shifts in the LSPR peak are reversible and the peak positions return to their pre-exposure values once the H2S is removed. The experirnental results strongly indicate that the protein based LSPR chip can be successfully used as a simple, fast, sensitive and quantitative sensor for H2S detection.

The hydrophobic cluster on the surface of protein is the key structural basis for the SDS-resistance of chondroitinase VhChlABC

The application of chondroitinase requires consideration of the complex microenvironment of the target.Our previous research reported a marine-derived sodium dodecyl sulfate(SDS)-resistant chondroitinase VhChlABC.This study further investigated the mechanism of VhChlABC resistance to SDS.Focusing on the hydrophobic cluster on its strong hydrophilic surface,it was found that the reduction of hydrophobicity of surface residues Ala181,Met182,Met183,Ala184,Val185,and Ile305 significantly reduced the SDS resistance and stability.Molecular dynamics(MD)simulation and molecular docking analysis showed that I305G had more conformational flexibility around residue 305 than wild type(WT),which was more conducive to SDS insertion and binding.The affinity of A181G,M182A,M183A,V185A and I305G to SDS was significantly higher than that of WT.In conclusion,the surface hydrophobic microenvironment composed of six residues was the structural basis for SDS resistance.This feature could prevent the binding of SDS and the destruction of hydrophobic packaging by increasing the rigid conformation of protein and reducing the binding force of SDS-protein.The study provides a new idea for the rational design of SDS-resistant proteins and may further promote chondroitinase research in the targeted therapy of lung diseases under the pressure of pulmonary surfactant.

Study of transactivating effect of pre-S2 protein of hepatitis B virus and cloning of genes transactivated by pre-S2 protein with suppression subtractive hybridization

AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization （SSH） technique, and to pave the way for elucidating the pathogenesis of HBV infection. METHODS： pcDNA3.1（-）-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 （-）-pre-S21pSV-lacZ and empty pcDNA3.1（-）/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase （β-gal）. SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1（-）-pre-S2 and pcDNA3.1（-） empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS： The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1（-）-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 （-）-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid （P〈0.01）. The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positiveclones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein. CONCLUSION： The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transcluction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.

A strong adhesive block polymer coating for antifouling of large molecular weight protein

Some proteins secreted by microorganisms have large molecular weights. We report here an approach to prepare coating by multilayer polymers for antifouling of proteins, especially the proteins with a large molecular weight.Stainless steel was used as the model substrate. The substrate was first coated with a hybrid polymer film, which was formed by simultaneous hydrolytic polycondensation of 3-aminopropyltriethoxysilane and polymerization of dopamine(HPAPD). After grafting the macroinitiator 2-bromoisobutyryl bromide, the block polymer brushes PMMA-b-PHEMA were grafted. Three proteins were used to test protein adsorption and antifouling behavior of the coating, including recombinant green fluorescent(54 k Da), recombinant R-transaminase(2 × 90 k Da), and recombinant catalase(4 × 98 k Da). It is demonstrated that the block polymer brushes not only can prevent the adsorption of small molecular weight proteins, but also can significantly reduce the adsorption of the large molecular weight proteins.

Analysis of the induction of the myelin basic protein binding to the plasma membrane phospholipid monolayer

Myelin basic protein（MBP） is an essential structure involved in the generation of central nervous system（CNS）myelin.Myelin shape has been described as liquid crystal structure of biological membrane.The interactions of MBP with monolayers of different lipid compositions are responsible for the multi-lamellar structure and stability of myelin.In this paper,we have designed MBP-incorporated model lipid monolayers and studied the phase behavior of MBP adsorbed on the plasma membrane at the air/water interface by thermodynamic method and atomic force microscopy（AFM）.By analyzing the pressure–area（π–A） and pressure–time（π–T） isotherms,univariate linear regression equation was obtained.In addition,the elastic modulus,surface pressure increase,maximal insertion pressure,and synergy factor of monolayers were detected.These parameters can be used to modulate the monolayers binding of protein,and the results show that MBP has the strongest affinity for 1,2-dipalmitoyl-sn-glycero-3-phosphoserine（DPPS） monolayer,followed by DPPC/DPPS mixed and1,2-dipalmitoyl-sn-glycero-3-phospho-choline（DPPC） monolayers via electrostatic and hydrophobic interactions.AFM images of DPPS and DPPC/DPPS mixed monolayers in the presence of MBP（5 n M） show a phase separation texture at the surface pressure of 20 m N/m and the incorporation of MBP put into the DPPC monolayers has exerted a significant effect on the domain structure.MBP is not an integral membrane protein but,due to its positive charge,interacts with the lipid head groups and stabilizes the membranes.The interaction between MBP and phospholipid membrane to determine the nervous system of the disease has a good biophysical significance and medical value.

Nucleos(t)ide analogues causes HBV S gene mutations and carcinogenesis

BACKGROUND： The long-term use of nudeos（t）ide analogues causes drug resistance and mutations in the HBV reverse tran- scriptase （RT） region of the polymerase gene. The RT region overlaps the HBV surface gene （S gene） and therefore, the mutations in the RT region simultaneously modify S gene sequence. Certain mutations in the RT region bring about truncated S proteins because the corresponding changed S gene encodes a stop codon which results in the loss of a large portion of the C-terminal hydrophobic region of HBV surface protein. The rtA181T/sW172＊, rtM204I/sW196＊ and rtV191I/sW182＊ are the most frequently reported drug-resistant mutations with C-terminal truncation, these mutations have oncogenic potential. DATA SOURCES： PubMed and Web of Science were searched using terms： ＂hepatitis B virus＂, ＂HBV drug resistance mutation＂ ＂HBV surface protein＂ ＂HBV truncation＂, ＂hepatocellular carcinoma＂, ＂rtA181T/sW172＊＂, ＂rtM204I/sW196＊＂, ＂rtV191I/sW182＊＂, and relevant articles published in English in the past decades were reviewed. RESULTS： The rtA181T/sW172＊ and rtV191I/sW182＊ mutants occurred more frequently than the rtM204I/sW196＊ mutant both in chronic hepatitis B patients and the HBV-related hepatocellular carcinoma tissues. Although these mutations occur naturally, nudeos（t）ide analogues therapy is the main driving force. These mutations may exist alone or coexist with other HBV mutations. All these three mutants impair the virion secretion and result in HBV surface protein retention and serum HBV DNA level reduction. These mutations possess potential carcinogenic properties. The three mutations are resistant to more than one nucleos（t）ide analogue and therefore, it is difficult to treat the patients with the truncated mutations.CONCLUSIONS： Nucleos（t）ide analogues induce drug resistance and HBV S gene truncated mutations. These mutations have potential carcinogenesis.

An endoparasitoid uses its egg surface proteins to regulate its host immune response

With proteomic analysis,we identified 379 egg surface proteins from an endoparasitoid,Cotesia chilonis.Proteins containing conserved enzymatic domains constitute a large proportion of egg surface components.Some proteins,such as superoxidase dismutase,homolog of C.rubecula 32-kDa protein,and immunoevasive protein-2A,are classical parasitism factors that have known functions in host immunity regulation.Melanization assays revealed that a novel egg surface protein,C.chilonis egg surface serpin domain-containing protein had the same function as a C.chilonis venom serpin,as both suppressed host melanization in a dose-dependent manner.C.chilonis egg surface serpin domain-containing protein is mainly transcribed in C.chilonis oocytes with follicular cells,and it is located on both the anterior and posterior sides of the mature egg surface.Additionally,we used LC-MS/MS to identify 586 binding proteins sourced from C.suppressalis plasma located on the eggshell surface of C.chilonis,which included some immunity-related proteins.These results not only indicate that C.chilonis uses its egg surface proteins to reduce the immune response of its host but also imply that endoparasitoid egg surface proteins might be a new parasitism factor involved in host immune regulation.

EXPRESSION OF Fas LIGAND IN HUMAN COLON CANCER CELL LINES

Objective: To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

An Approximated Voxel Approach for the Identification and Modelling of Ligand-Binding Sites

Most protein-ligand interactions take place on surfaces and include but not limited to factors such as chemical composition, hydrophobicity, electronegavitiy and shape complementarity. Past studies showed that protein-protein interactions occur on comparatively fiat regions whereas protein-ligand bindings involve crevices. In the search for such sites various approaches have been designed and developed each of which is algorithmically unique. The use of grid units or voxels has been demonstrated in early studies with relatively good results obtained. We present here an approximated approach comprising of the use of voxels and computer vision methods in the search for ligand-binding areas. Each test protein is modelled and analysed in 2D with all corresponding residues graphically presented for successfully identified sites. The study was carried out on 2 sets of proteins： FK506-bound proteins and heme-bound proteins with promising results obtained for all test cases.

A new photolabeling probe for efficient enrichment and deep profiling of cell surface membrane proteome by mass spectrometry

The cell surface membrane proteome is a class of proteins encoded by ~25% of all protein-coding genes in living organisms and plays a key role in mediating communication between the cells and their surrounding environment. However, most cell surface membrane proteins(CSMPs) are naturally expressed at very low levels compared with intracellular proteins. The difficulties in their purification with high specificity further hinder the understanding of their structure and function. In this study, we developed a new photolabeling probe to achieve efficient tagging and facile enrichment of the CSMPs. The probe is composed of a lipid tail for cell surface localization, a polyethylene glycol(PEG) spacer for increased water solubility, two 4-(N-maleimido)benzophenone(MBP) groups for UV-active tagging of the CSMPs, and a biotin tag for subsequent isolation. Application of this photolabeling probe resulted in the successful enrichment and identification of 3098 annotated CSMPs in HT22 cells with close to 70% selectivity. The proposed photolabeling probe and enrichment strategy were demonstrated to be a powerful method for deep cell surface proteome profiling, representing one of the largest groups of current drug targets.

Multiomics approaches to uncover endometrial receptivity in embryo implantation:a mini-review

Successful implantation requires concerted interactions during the apposition,adhesion,and invasion of the embryo into a receptive endometrium.However,the embryo implantation rate for assisted reproduction remains low despite the transfer of good quality embryos.Changes in endometrial transcriptomics,proteomics,lipidomics,and even microbiota all play important roles in embryo implantation.Specifically,the expression of steroid hormone-regulated adhesive and anti-adhesive molecules during the embryo implantation window is becoming an area of increasingly intense research.This review(a)summarizes the different molecules expressed in the receptive endometrium and(b)proposes the use of surface protein markers to predict pregnancy outcomes from assisted reproduction.

Synthesis of surface imprinted polymers based on wrinkled flower-like magnetic graphene microspheres with favorable recognition ability for BSA

Nowadays,the employing of molecular imprinting technique in the analysis and separation of proteins from complex biological samples has been widely favored by researchers.To enrich the types of surface protein imprinted materials and expand the application fields of graphene materials,novel surface molecular imprinted polymers(MIPs)based on magnetic graphene microspheres Fe_(3)O_(4)@r GO@MIPs are first synthesized in this paper.Fe_(3)O_(4)@r GO@MIPs are prepared by oxidative self-polymerization of dopamine on the surface of magnetic graphene(Fe_(3)O_(4)@r GO)composite microspheres.Bovine serum albumin(BSA)is selected as protein template.Fe_(3)O_(4)@r GO microspheres with wrinkled flower-like structure are obtained by compounding Fe_(3)O_(4)and graphene oxide in an appropriate ratio via the method of high-temperature reduction self-assembly.The microspheres exhibit promising dispersibility,high external surface area,rich pore structure,and sufficient magnetic properties.These advantages not only prevent the agglomeration of imprinted microspheres in the aqueous phase,which is conducive to contact and static adsorption,but also increase the amount of protein imprinting.Additionally,sufficient magnetic properties ensure fast and effective separation of the adsorbents.While the adsorption capacity is increased,the separation procedure becomes simple.The binding capacity of Fe_(3)O_(4)@r GO@MIPs for BSA can reach 317.58 mg/g within 60 min,and the imprinting factor(IF)is 4.24.More importantly,Fe_(3)O_(4)@r GO@MIPs can specifically recognize the target BSA from the mixed proteins and the actual sample.There is no significant decrease in the adsorption amount,IF,and magnetic properties after eight runs.It is promising to be used in the separation of proteins from the actual biological samples.

Construction of Nanophase Novel Coatings-Based Titanium for the Enhancement of Protein Adsorption

In the recent years,biological nanostructures coatings have been incorporated into orthopedic and dental implants in order to accelerate osseointegration and reducing surgical restrictions.In the present work,chemical etching,anodization and metal doping surface modification methods were integrated in one strategy to fabricate innovative titanium surfaces denominated by titanium nanoporous,anodized titanium nanoporous,silver-anodized titanium nanoporous and gold-anodized titanium nanoporous.The stability properties of nanostructures-coated surfaces were elucidated using electrochemical impedance spectroscopy（EIS） after 7 days of immersion in simulated biological fluids.Morphology and chemical compositions of new surfaces were characterized by scanning electron microscope and energy-dispersive X-ray analysis.The EIS results and data fitting to the electrical equivalent circuit model demonstrated the influence of adsorption of bovine serum albumin on new surfaces as a function of protein concentration.Adsorption process was described by the very well-known model of the Langmuir adsorption isotherm.The thermodynamic parameter DGADS（-50 to 59 kJ mol^（-1）） is calculated,which supports the instantaneous adsorption of protein from biological fluids to new surfaces and refers to their good biocompatibility.Ultimately,this study explores new surface strategy to gain new implants as a means of improving clinical outcomes of patients undergoing orthopedic surgery.