Evaluation of Six Recombinant Proteins for Serological Diagnosis of Lyme Borreliosis in China

Objective In this study, we evaluated the diagnostic efficiency of six recombinant proteins for the serodiagnosis of Lyme borreliosis （LB） and screened out the appropriate antigens to support the production of a Chinese clinical ELISA （enzyme-linked immunosorbent assay） kit for LB. Methods Six recombinant antigens, Fla B.g, OspC B.a, OspC B.g, P39 B.g, P83 B.g, and VlsE B.a, were used for ELISA to detect serum antibodies in LB, syphilis, and healthy controls. The ELISA results were used to generate receiver operating characteristic （ROC） curves, and the sensitivity and specificity of each protein was evaluated. All recombinant proteins were evaluated and screened by using logistic regression models. Results Two IgG （VISE and OspC B.g） and two IgM （OspC B.g and OspC B.a） antigens were left by the logistic regression model screened. VIsE had the highest specificity for syphilis samples in the IgG test （87.7%, P〈0.05）. OspC B.g had the highest diagnostic value in the IgM test （AUC=0.871）. Interactive effects between OspC B.a and Fla B.g could reduce the specificity of the ELISA. Conclusion Three recombinant antigens, OspC B.g, OspC B.a, and VisE B.a, were useful for ELISAs of LB. Additionally, the interaction between OspC B.a and Fla B.g should be examined in future research.

Safety of Human Recombinant Proteins

Recombinant human proteins play an important role in therapy, especially in stimulating the hematopoiesis after chemotherapy, e. g., erythropoietin and colony stimulating factors,while several promising candidates such as IL-6, IL-12, thrombopoietin and others are in clinical development. Since the recombinant proteins are copies of endogenous proteins, it was assumed that they would be well tolerated. While this assumption is correct for some, other proteins proved to be less well tolerated. Therefore, preclinical safety assessment of these pro teins is necessary. Based on the experience with several proteins, some guidance for the safety assessment can be given. Furthermore, data are presented demonstrating that preclinical toxi city studies may have a predictive value for man. Limitations of the classical approach of safety tests and new concepts are discussed

Susceptibility to AcMNPV and Expression of Recombinant Proteins by a Novel Cell Clone Derived from a Trichoplusia ni QAU-BTI-Tn9-4s Cell Line

It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines.But the characteristics of the cell line do not always remain stable and may change upon continuous passage.Recently an alphanodavirus,named Tn5 Cell Line Virus (or TNCL Virus),was identified in High Five cells in particular.Therefore,we established a new cell line,QB-Tn9-4s,from Trichoplusia ni,which was determined to be free of TNCL virus by RT-PCR analysis.In this paper,we describe the development of a novel cell clone,QB-CL-B,from a low passage QB-Tn9-4s cell line and report its susceptibility to AcMNPV,and the level of recombinant protein production.This cell clone was similar to its parental cells QB-Tn9-4s and Tn5B1-4 cells in morphology and growth rate;although it also showed approximately the same responses to AcMNPV infection and production of occlusion bodies,there were higher levels of recombinant protein production in comparison to QB-Tn9-4s (parental cells) and High5 cells.

Engineering Saccharomyces cerevisiae for efficient production of recombinant proteins

Saccharomyces cerevisiae is an excellent microbial cell factory for producing valuable recombinant proteins because of its fast growth rate,robustness,biosafety,ease of operability via mature genomic modification technologies,and the presence of a conserved post-translational modification pathway among eukaryotic organisms.However,meeting industrial and market requirements with the current low microbial production of recombinant proteins can be challenging.To address this issue,numerous efforts have been made to enhance the ability of yeast cell factories to efficiently produce proteins.In this review,we provide an overview of recent advances in S.cerevisiae engineering to improve recombinant protein production.This review focuses on the strategies that enhance protein production by regulating transcription through promoter engineering,codon optimization,and expression system optimization.Additionally,we describe modifications to the secretory pathway,including engineered protein translocation,protein folding,glycosylation modification,and vesicle trafficking.Furthermore,we discuss global metabolic pathway optimization and other relevant strategies,such as the disruption of protein degradation,cell wall engineering,and random mutagenesis.Finally,we provide an outlook on the developmental trends in this field,offering insights into future directions for improving recombinant protein production in S.cerevisiae.

Recombinant human zona pellucida proteins ZP1, ZP2 and ZP3 co-expressed in a human cell line

Aim: To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests. Methods: The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed. Results: RhZP2 and rhZP3 were secreted into the culture medium, whereas rhZPl was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZPl was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed. Conclusion: RhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZPl from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.

A suspended cell line from Trichoplusia ni （Lepidoptera）： Characterization and expression of recombinant proteins

A suspended cell line from Trichoplusia ni embryos was established, and its susceptibility to Autographa californica multiple nuclear polyhedrosis virus （AcMNPV） infection was investigated. This cell line had characteristics distinct from the BTI-Tn5B 1- 4 cell line （Tn5B 1-4） from T. ni in growth, and showed approximately the same responses to AcMNPV infection, production of occlusion bodies, and levels of recombinant protein expression. No clumps were observed at maximum cell density at late-log phase in shake-flask or T-flask cultures, and thus the cells represent a useful new contribution for baculovirus research. The cells consist of two major morphological types： approximately 70% spindle-shaped cells and 30% round cells. The cell line was highly susceptible to virus infection and produced around 107 AcMNPV occlusion bodies per cell, on average. Production of β-galactosidase and secreted alkaline phosphatase was high with 3.97 ± 0.13 × 10^4 IU/mL and 3.48 ± 0.40IU/mL, respectively. This cell line may be applicable for studies of scale-up production of viruses or baculovirus-insect cell expression. We also believe the new line can be a source for cell clones with higher production of virus and recombinant proteins compared to the parent or other existing cell lines such as Tn5B 1-4.

Active Peptides and Motifs within Collagen and Other ECM Proteins

Collagens are the most abundant proteins in mammals and form an extracellular matrix (ECM) with other components as the structural support of muscle, skin, corneas and blood vessels etc. Other than providing structural support, the ECM exhibits active communication with cells and influences many cellular processes including migration, wound healing, differentiation and cancer metastasis. Though collagen proteins contain highly repetitive primary sequences and defined tertiary structures, more and more studies have shown that many short peptides/motifs within collagen proteins play key roles in various biological processes. These short sequences are effective within triple helical structures or independently as stand-alone molecules resulting from proteolytic degradation. Besides endogenous ECM-derived peptides, many more functional peptides have been produced by tissue processing, chemical synthesis, and recombinant protein production. In this review, we summarize different peptides/motifs identified in collagen and other ECM proteins and discuss their potential for medical, personal care, and cosmetics applications.

Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.

Prokaryotical expression of structural and non-structural proteins of hepatitis G virus

AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.

Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

Suppression of colorectal tumorigenesis by recombinant Bacteroides fragilis enterotoxin-2 in vivo

AIM To evaluate the impact of recombinant Bacteroides fragilis enterotoxin-2 (BFT-2, or Fragilysin) on colorectal tumorigenesis in mice induced by azoxymethane/dextran sulfate sodium (AOM/DSS). METHODS Recombinant proBFT-2 was expressed in Escherichia coli strain Rosetta (DE3) and BFT-2 was obtained and tested for its biological activity via colorectal adenocarcinoma cell strains SW-480. Seventy C57BL/6J mice were randomly divided into a blank (BC; n = 10), model (AD; n = 20), model + low-dose toxin (ADLT; n = 20, 10 mu g), and a model + high-dose toxin (ADHT; n = 20, 20 mu g) group. Mice weight, tumor formation and pathology were analyzed. Immunohistochemistry determined Ki-67 and Caspase-3 expression in normal and tumor tissues of colorectal mucosa. RESULTS Recombinant BFT-2 was successfully obtained, along with its biological activity. The most obvious weight loss occurred in the AD group compared with the ADLT group (21.82 +/- 0.68 vs 23.23 +/- 0.91, P < 0.05) and the ADHT group (21.82 +/- 0.68 vs 23.57 +/- 1.06, P < 0.05). More tumors were found in the AD group than in the ADLT and ADHT groups (19.75 +/- 3.30 vs 6.50 +/- 1.73, P < 0.05; 19.75 +/- 3.30 vs 6.00 +/- 2.16, P < 0.05). Pathology showed that 12 mice had adenocarcinoma and 6 cases had adenoma in the AD group. Five mice had adenocarcinoma and 15 had adenoma in the ADLT group. Four mice had adenocarcinoma and 16 had adenoma in the ADHT group. The incidence of colorectal adenocarcinoma in both the ADHT group and the ADHT group was reduced compared to that in the AD group (P < 0.05, P < 0.05). The positive rate of Ki-67 in the ADLT group and the ADHT group was 50% and 40%, respectively, both of which were lower than that found in the AD group (94.44%, P < 0.05, P < 0.05). Caspase-3 expression in the ADLT group and the ADHT group was 45% and 55%, both of which were higher than that found in the BC group (16.67%, P < 0.05, P < 0.05). CONCLUSION Oral administration with lower-dose biologically active recombinant BFT-2 inhibited colorectal tumorigenesis in mice.

Construction and Expression of Tp0453 Recombinant Protein of Treponema pallidum and Development of Indirect ELISA for Diagnosinq Syphilis

Objectives： To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods： The immuno-dominant epitope of Tp0453 gene was amplified from the complete genome of T.pallidum by polymerase chain reactions （PCR）, subcloned into the expression vector Pqe32 to generate recombinant plasmid Pqe32/Tp0453, and was then expressed in E. coli M15. The fusion protein was purified with Ni-NTA affinity purification. Indirect ELISA was developed to detect human serum IgG antibody to T. pallidum. Results： The recombinant Tp0453 protein was successfully expressed and purified. The recombinant protein had a molecular weight of approximately 32KDa.Indirect ELISA to the recombinant protein was developed.Sixty control sera were tested by ELISA and yielded a sensitivity of 100% （30/30） and a specificity of 100% （30/30）. While testing for T. pallidum in human sera, the sensitivity and specificity of ELISA were 96.8% and 100%, respectively, when compared with TPPA test results. The concordance of results between the ELISA test and the TPPA test was 98.2%. Conclusion： The recombinant Tp0453 outer membrane protein elicited a strong immunoreaction to anti-T.pallidum IgG antibody and has great potential use in ELISA for the serodiagnosis of syphilis.

Green Fluorescent Protein Recombinant Nisin as a Probe for Detection of Gram-Positive Bacteria

A great amount of foodborne pathogens were Gram-positive (G+) bacteria, a threat to public health. In this study, considering the binding ability of nisin towards G+ bacteria and the stable fluorescent ability of EGFP protein, a fluorescent nisin–EGFP protein probe was constructed by a gene engineering method. Nisin and EGFP were used as the receptor and fluorophore, respectively, to detect G+ bacteria. The nisin and egfp gene were amplified separately according to the sequence published in GenBank using unique primers. The two genes were cloned into a pET-28b(+) vector resulting in a pET-28b(+)–nisin–egfp vector. The vector was transferred into Escherichia coli (E. coli) BL21 (DE3) for expression. The expressed protein was extracted, purified by a Ni–NTA column, and then tested by the SDS-PAGE method to confirm its molecular weight. Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus (S. aureus), and Micrococcus luteus (M. luteus) were used as the representations of G+ bacteria. E. coli O157, representing the gram-negative (G−) bacteria, was used as a negative control. The binding specificity of the recombinant protein was performed on two types of bacteria and then detected through fluorescent microscopy. The results indicated that the nisin–EGFP probe could detect G+ bacteria at 108CFU/mL. © 2017, Tianjin University and Springer-Verlag Berlin Heidelberg.

The expression and antigenicity identification of recombinant rat TGF-β1 in bacteria

In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.

Recombinant human bone morphogenetic protein-7 expressed from CHO cells possessing the activity of bone-induced in vitro

Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.

Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene （ompW） from E parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by E parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs

African swine fever(ASF),caused by the African swine fever virus(ASFV),is a devastating disease of domestic and wild pigs.There is no effective vaccine,and the control of the disease relies mainly on surveillance and early detection of infected pigs.Previously,serological assays,such as ELISA,have been developed mainly based on recombinant structural viral proteins of ASFV,including p72,p54,and p30.However,the antibodies against these proteins do not provide efficient protection against ASFV infection in pigs.Therefore,new serological assays that can be applied for clinical diagnosis and evaluating serological immune response in vaccinated pigs are still required.In this study,we expressed and purified a recombinant p B602 L protein.The purified p B602 L protein was then used as an antigen to develop an indirect ELISA assay.This assay has no cross-reaction with the anti-sera against the 15 most common pig pathogens in China,such as classical swine fever virus,pseudorabies virus,and porcine parvovirus.This assay and a commercial ELISA kit were then used to detect 60 field pig serum samples,including an unknown number of antiASFV sera.The coincidence of the two assays was 95%.Furthermore,the p B602 L-based ELISA was employed to test the antibody responses to the seven-gene-deleted ASFV strain HLJ/18-7 GD in pigs.The results showed that the antibody levels in all vaccinated pigs,starting from the 10 th day post-inoculation,have increased continuously during the observation period of 45 days.Our results indicate that this p B602 L-based indirect ELISA assay can be employed potentially in the field of ASFV diagnosis.

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c(Hsp70_(359–610)) fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70(mHsp70) is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte(CTL) response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2(M2e), was fused to the C-terminus of Mycobacterium tuberculosis Hsp70(Hsp70c), to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c(Hsp70359–610). And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.

Expression of fluorescent tagged recombinant erythroferrone protein

Objective: To produce fluorescent tagged recombinant erythroferrone protein(ERFE_eGFP) for laboratory investigations. Methods: Erythroferrone(ERFE) gene was fused to green fluorescent protein(eGFP) gene and cloned in a pSecTag2Hygro plasmid. The constructed plasmid was amplified in Escherichia coli DH5α and the eGFP-fused ERFE(ERFE_eGFP) protein was expressed in human embryonic kidney(HEK293T) cell line. Results: The plasmid constructed from colony C6 contained ERFE_eGFP with the correct restriction sizes of 4.2 kb and expressed secretory ERFE_eGFP fusion protein(approximately size of 75 kDa) in HEK293T cell line. Conclusions: ERFE_eGFP recombinant protein is successfully expressed as a secretory functional protein and could be sensitively detected using fluorometry. This fusion protein might benefit future applications for localization of cellular ERFE receptors and competitive immunoassay of ERFE concentration.