scholarly journals Specific T-cell Responses to CFP10, an Secreted Antigens of Mycobacterium Tuberculosis Protein, in Chinese HIV Positive Individuals

2013 ◽  
Vol 2 (1) ◽  
pp. 6-12
Author(s):  
Bin Zhang ◽  
Wen-hui Lun ◽  
Xing-wang Li ◽  
Qi Wang ◽  
Jun Cheng ◽  
...  
Keyword(s):  
T Cell ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Prokaryotic Expression ◽  
Expression Vector ◽  
T Cell Responses ◽  
Hiv Positive ◽  
E Coli ◽  
Cell Responses ◽  
Prokaryotic Expression Vector

Abstract Objective To construct prokaryotic expression vector of CFP-10 gene, and obtain recombinant protein, and the recombinant CFP-10 protein was taken as stimulus to detect specific T cell responses, to set up a method to faciliate to detect potential TB infection in China. Methods CFP-10 was cloned into inducible prokaryotic expression vector pET-32a (+) and transfected into E. coli BL21 (DE3). After IPTG induction, the product were verified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot hybridization were carried out to verify the antigenicity; the recombinant CFP-10 protein was taken as stimulus to detect specific T cell responses in HIV (+) persons with or without clinical manifestation of TB diseases, and HIV (-) controls with or without TB diseases. Results The CFP-10 recombinant protein exsited in the form of inclusion body and accounted for 94% in total bacterial protein of E. coli and the molecular weight is 31 kD; Western blot confirmed the recombinant proteins had high antigenicity; our in-house ELISpot-IFN-γ assay with recombinant antigen derived from CFP-10 proteins showed significant higher frequencies in TB patients with or without HIV infection than that in the healthy controls and only HIV (+) group. Conclusions The recombinant CFP-10 genes can be expressed successfully in prokaryotic expression system of E. coli and recombinant proteins with high antigenicity were obtained, which will set foundation for further study on their immunogenicity and bioinformatics. Our results proved that it is indeed true that some HIV positive patient have high frequencies of TB specific T cell responses, which maybe a clue to find latent TB infection in this population.

Hookworm recombinant protein promotes regulatory T cell responses that suppress experimental asthma

2016 ◽  
Vol 8 (362) ◽  
pp. 362ra143-362ra143 ◽  
Author(s):  
S. Navarro ◽  
D. A. Pickering ◽  
I. B. Ferreira ◽  
L. Jones ◽  
S. Ryan ◽  
...  
Keyword(s):  
T Cell ◽  
Recombinant Protein ◽  
Regulatory T Cell ◽  
T Cell Responses ◽  
Cell Responses

Construction of Prokaryotic Expression Vector Containing Flocculation Gene FLO5 and Expression of FLO5 in E. coli

2013 ◽  
Vol 310 ◽  
pp. 157-161 ◽  
Author(s):  
Jie Xue ◽  
Wen Qiang Wei ◽  
Dong Yan Zhang ◽  
Yong Li Li ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Prokaryotic Expression ◽  
Genomic Dna ◽  
Expression Vector ◽  
Gene Fragment ◽  
Form Expression ◽  
Protein Fragments ◽  
E Coli ◽  
Sds Page ◽  
Prokaryotic Expression Vector

FLO5 has been identified as a dominant flocculation gene. The goal of this study is to clone the FLO5 gene from Saccharomyces cerevisiae and express it in E. coli. In this study, the FLO5 gene amplified by PCR from S. cerevisiae was cloned into prokaryotic expression vector pET-28a to form expression vector pET28a-FLO5, finally, transferred into E.coli BL21. Methods: FLO5 gene was amplified by PCR from genomic DNA extracted from Saccharomyces cerevisiae. The amplified FLO5 gene fragment was then recombined with clone vector pMD18-T to form clone vector pMD18-T-FLO5 amplified in E.coli JM109. After confirmed with sequencing, FLO5 fragment cut out from pMD18-T-FLO5 by enzyme EcoRI and NotI was recombined into expression vector pET-28a to form vector pET28a-FLO5. Vector pET28a-FLO5 was then transferred into E. coli BL21 and protein FLO5 was expressed in E. coli BL21 by the induction with IPTG. Expressed protein fragments separated by SDS-PAGE showed a band with the size of protein FLO5 suggesting the expression of gene FLO5. with the expected This study will lay the foundation for further research in studying flocculating effect of exogenous protein expressed by genetic engineering and making new flocculating agent through recombinant engineering.

scholarly journals A recombinant protein containing influenza viral conserved epitopes and superantigen induces broad-spectrum protection

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Yansheng Li ◽  
Mingkai Xu ◽  
Yongqiang Li ◽  
Wu Gu ◽  
Gulinare Halimu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
T Cell ◽  
Recombinant Protein ◽  
Broad Spectrum ◽  
T Cell Responses ◽  
H1n1 Influenza ◽  
Influenza Viruses ◽  
Lung Damage ◽  
Universal Vaccine ◽  
Cell Responses

Influenza pandemics pose public health threats annually for lacking vaccine which provides cross-protection against novel and emerging influenza viruses. Combining conserved antigens that induce cross-protective antibody responses with epitopes that activate cross-protective T cell responses might be an attractive strategy for developing a universal vaccine. In this study, we constructed a recombinant protein named NMHC which consist of influenza viral conserved epitopes and a superantigen fragment. NMHC promoted the maturation of bone marrow-derived dendritic cells and induced CD4+ T cells to differentiate into Th1, Th2, and Th17 subtypes. Mice vaccinated with NMHC produced high levels of immunoglobulins that cross-bound to HA fragments from six influenza virus subtypes with high antibody titers. Anti-NMHC serum showed potent hemagglutinin inhibition effects to highly divergent group 1 (H1 subtype) and group 2 (H3 subtype) influenza virus strains. Furthermore, purified anti-NMHC antibodies bound to multiple HAs with high affinities. NMHC vaccination effectively protected mice from infection and lung damage when exposed to two subtypes of H1N1 influenza virus. Moreover, NMHC vaccination elicited CD4+ and CD8+ T cell responses that cleared the virus from infected tissues and prevented virus spread. In conclusion, this study provides proof of concept that NMHC vaccination triggers B and T cell immune responses against multiple influenza virus infections. Therefore, NMHC might be a candidate universal broad-spectrum vaccine for the prevention and treatment of multiple influenza viruses.

Construction and expression of a prokaryotic expression vector for the goat sry gene

2019 ◽  
Author(s):  
Xiao Wang ◽  
Guang-Xin E ◽  
Shu-Zhu Cheng ◽  
Wei-Wei Ni ◽  
Yue-Hui Ma ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Prokaryotic Expression ◽  
Expression Vector ◽  
Target Gene ◽  
Rabbit Antiserum ◽  
Rabbit Serum ◽  
Molecular Sexing ◽  
Coding Region ◽  
Sry Gene ◽  
Prokaryotic Expression Vector

Goats are economically important animals in the world, and their sex is an important factor in their economic efficiency. Reconstruction of a goat SRY gene expression vector can lay a foundation for studying the immunogenicity and sex determination of SRY protein at the molecular level. In this study, the coding region of the goat SRY gene was used as the target gene fragment for synthesis and optimization, and the cloning vector was used as a template to amplify the target gene and finally ligated to the expression vector pET-SUMO. The recombinant plasmid was then verified by the double restriction enzyme method and transformed into Escherichia coli (DE3). After the induction of expression by Isopropyl â-D-Thiogalactoside (IPTG), the cells were lysed, and SDS-PAGE electrophoresis was performed to observe the expression of the recombinant protein. Additionally, the immunological activity of the recombinant protein was assessed. The target gene was successfully ligated into the prokaryotic expression vector pET-28a; additionally, the prokaryotic expression plasmid pET-SUMO was successfully constructed, and the SRY antigen protein (42 kDa) was expressed. The titer of the rabbit antiserum (PAI-1608012-1) was more than 1:50000, as measured by ELISA, which demonstrated that the titer and the sensitivity of the rabbit serum reached the expected levels. In this study, the prokaryotic expression vector pET-SUMO was successfully constructed. The recombinant protein has high immunopotency and immunoreactivity, which lays a foundation for the preparation of antibodies and the molecular sexing of goats in the future.

scholarly journals Broadening CD4+ and CD8+ T Cell Responses against Hepatitis C Virus by Vaccination with NS3 Overlapping Peptide Panels in Cross-Priming Liposomes

2017 ◽  
Vol 91 (14) ◽  
Author(s):  
Jonathan Filskov ◽  
Marianne Mikkelsen ◽  
Paul R. Hansen ◽  
Jan P. Christensen ◽  
Allan R. Thomsen ◽  
...  
Keyword(s):  
T Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Recombinant Protein ◽  
T Cell Responses ◽  
T Cell Response ◽  
T Cell Epitopes ◽  
Cell Responses ◽  
Vaccine Approach

ABSTRACT Despite the introduction of effective drugs to treat patients with chronic hepatitis C virus (HCV) infection, a vaccine would be the only means to substantially reduce the worldwide disease burden. An incomplete understanding of how HCV interacts with its human host and evades immune surveillance has hampered vaccine development. It is generally accepted that in infected individuals, a narrow repertoire of exhausted T cells is a hallmark of persistent infection, whereas broad, vigorous CD4+ and CD8+ T cell responses are associated with control of acute hepatitis C. We employed a vaccine approach based on a mixture of peptides (pepmix) spanning the entire sequence of HCV nonstructural protein 3 (NS3) in cross-priming cationic liposomes (CAF09) to facilitate a versatile presentation of all possible T cell epitopes, regardless of the HLA background of the vaccine recipient. Here, we demonstrate that vaccination of mice with NS3 pepmix broadens the repertoire of epitope-specific T cells compared to the corresponding recombinant protein (rNS3). Moreover, vaccination with rNS3 induced only CD4+ T cells, whereas the NS3 pepmix induced a far more vigorous CD4+ T cell response and was as potent a CD8+ T cell inducer as an adenovirus-vectored vaccine expressing NS3. Importantly, the cellular responses are dominated by multifunctional T cells, such as gamma interferon-positive (IFN-γ+) tumor necrosis factor alpha-positive (TNF-α+) coproducers, and displayed cytotoxic capacity in mice. In conclusion, we present a novel vaccine approach against HCV, inducing a broadened T cell response targeting both immunodominant and potential subdominant epitopes, which may be key elements to counter T cell exhaustion and prevent chronicity. IMPORTANCE With at least 700,000 annual deaths, development of a vaccine against hepatitis C virus (HCV) has high priority, but the tremendous ability of the virus to dodge the human immune system poses great challenges. Furthermore, many chronic infections, including HCV infection, have a remarkable ability to drive initially strong CD4+ and CD8+ T cell responses against dominant epitopes toward an exhausted, dysfunctional state. Thus, new and innovative vaccine approaches to control HCV should be evaluated. Here, we report on a novel peptide-based nanoparticle vaccine strategy (NS3 pepmix) aimed at generating T cell immunity against potential subdominant T cell epitopes that are not efficiently targeted by vaccination with full-length recombinant protein (rNS3) or infection with HCV. As proof of concept, we found that NS3 pepmix excels in broadening the repertoire of epitope-specific, multifunctional, and cytotoxic CD4+ and CD8+ T cells compared to vaccination with rNS3, which generated only CD4+ T cell responses.

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