Fungi immunoregulatory proteins family is effective in immunological regulation and anti-tumor. We used Pichia pastoris expression system for recombinant expression of Lz-8, an immunomodulatory protein isolated from fruiting body of Ganoderma lucidum. The Gs115 (mut+) strains of P. pastoris was used as host cells. PCR and sequencing of DNA showed that Lz-8 cDNA was successfully integrated into the P. pastoris genome. Electrophoresis (SDS-PAGE), matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and immunological techniques were used to identify recombinant Lz-8 (rLz-8). Lz-8 expressed in Escherichia coli, the Pichia system requires further optimization to obtain more active fungi immunomodulatory protein. Lz-8 was expressed in P. pastoris successfully, and polyacrylamide gel electrophoresis in the presence of SDS-PAGE gave a single band with an apparent Mr=14,000 D. MALDI-TOF-MS also showed that molecular weight of rLz-8 was 12,722 D. Aggregation was observed from sheep red blood cells in the presence of purified rLz-8 within the concentration range of 12.5-50 microg/mL. However, no aggregation was seen at concentration greater than 50 microg/mL for any type of human red blood cell. The dose at 0.5 mg/kg of rLz-8 induced macrophage cytophagocytesis, and set interferon as control at 0.5 mg/kg. These results suggested that active and stable rLz-8 was obtained in P. pastoris expression system.
Fungal Proteins ; biosynthesis ; genetics ; immunology ; Immunocompetence ; immunology ; Macrophages ; immunology ; Phagocytosis ; drug effects ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

Escherichia coli ; genetics ; Humans ; Recombinant Fusion Proteins ; immunology ; Viral Nonstructural Proteins ; genetics ; immunology

The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.
Cloning, Molecular ; Nucleocapsid Proteins ; biosynthesis ; genetics ; immunology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; SARS Virus ; genetics

Cloning, Molecular ; Escherichia coli ; genetics ; Hepacivirus ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology

Foot-and-mouth disease virus (FMDV) is the aetiological angent of a highly contagious viral disease. The complete gene encoding the structural protein of FMDV (P1) was subcloned into expression vector pGEX-KG, resulting in the fusion expression plasmid pKG-P1. After transformed into E. coli BL21(DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-P1 fusion protein was expressed in high level. The molecular weight of the fusion protein wa 110kD and the expressed products were soluble. Western-blotting was performed to confirm that the expressed fusion protein could specifically react with antiserum against FMDV. The fusion proteins were further purified by GST purification kit and an indirect ELISA (P1-ELISA) based on the purified proteins was developed. Comparison between P1-ELISA and the standard indirect haemagglutinin assay showed the two methods had 87 per cent agreement by detecting 864 serum samples, indicating the purified P1 protein was specific as the antigen of indirect P1-ELISA.
Capsid Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease Virus ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Pseudorabies virus (PRV), an alpha-herpesvirus, has been used as a vector for live-viral animal vaccines. The recombinant PRV TK- / gE- / GP5+, which expressing GP5 of PRRSV, is developed based on the PRV genetic-depleting vaccine-virus strain, TK- / gE- /LacZ+. However, this strain stimulated poorly the vaccinated animals to produce neutralizing antibodies against PRRSV. In order to develop a booster specific immunized response of the PRV recombinant, the ORF5 gene of PRRSV TK- / gE- / LacZ+ was substituted by a modified ORF5 gene, ORF5m. The resultant recombinant PRV, TK- /gE- / GP5m+, was verified by PCR, Southern blotting and Western blotting. TK- / gE- / GP5m+ and TK- / gE- / GP5+ expressed GP5 proteins were inoculated into balb/c mice to evaluate their immunogenicity. The results demonstrated that the amount of neutralization antibodies and cell-immunity responses induced by TK- / gE- /GP5m+ against PRRSV were higher than that of TK- / gE- / GP5+. This study indicated that the new recombinant PRV expressing the modified GP5m protein is a candidate for the development of bivalent genetic engineering vaccines against PRRSV and PRV.
Animals ; Genetic Vectors ; Herpesvirus 1, Suid ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Porcine respiratory and reproductive syndrome virus ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Viral Envelope Proteins ; biosynthesis ; genetics ; Viral Vaccines ; genetics ; immunology

OBJECTIVETo clone the gene encoding nucleocapsid protein (NP) of hantavirus strain Z10 (HV-Z10), to construct its prokaryotic expression system as well as to establish a rNP-IgM direct capture ELISA based on HRP-labeled recombinant NP (rNP), in order to detect serum samples of patients suffering from hemorrhagic fever with renal syndrome (HFRS) and to evaluate the effects of detection.

METHODSGene encoding NP of strain HV-Z10 was amplified by PCR and then its prokaryotic expression system pET28a-Z10N-E. coli BL21DE3 was constructed, using routine genetic engineering method. SDS-PAGE was applied to measure the expression of rNP and ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product. Western blot assay was used to determine the specific immuno-reactivity of rNP while HRP-labeled rNP-IgM direct capture ELISA was established to detect the serum samples from 95 cases of confirmed HFRS patients. The detection effect was compared with that by routine HV-IgM indirect capture ELISA method.

RESULTSpET28a-Z10N-E. coli BL21DE3 was able to express rNP with high efficiency. The purified rNP only showed a single protein fragment in the gel after SDS-PAGE. HV-IgG could efficiently recognize rNP and hybridize with the recombinant protein. 94.73% (90/95) of HFRS patients' serum samples were positively confirmed by rNP-IgM direct capture ELISA, while a positive rate of 92.63% (88/95) in the same samples was confirmed by HV-IgM indirect capture ELISA. The distributions of A450 values of the serum samples detected by the two IgM capture ELISAs as well as the changes of the A450 mean values from several serum samples with different dilutions were similar.

CONCLUSIONWe successfully constructed a high efficient prokaryotic expression system of NP encoding gene of hantavirus strain HV-Z10. The rNP-IgM direct capture ELISA that established in this study could be used as a new serological test for HFRS diagnosis because of its simplicity, safety, with high sensitivity and specificity.

Blotting, Western ; Capsid Proteins ; genetics ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Immunoglobulin M ; immunology ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Viral Core Proteins ; genetics ; immunology ; metabolism

In the study, a gene encoding Tat protein N terminal 1- 21 amino acid residues-deleted mutant (Tat22-101) was amplified by PCR from a full length Tat gene of human immunodeficiency virus type 1, and the prokaryotic expression plasmid pET32a-Tat22-101 was constructed. After identification by digestion with endonucleases and sequencing, the recombinant plasmid pET32a-Tat22-101 was transformed into E. coli BL21(DE3) and expressed with IPTG induction. The mutant fusion protein with deleted Tat N terminal was purified by an affinity chromatography column Ni(2+)-NTA and subsequently identified by SDS-PAGE and Western blotting. The results showed that the molecular weight of the mutant protein was approximately 26.9kD. Furthermore, BALB/c mice were immunized with the mutant protein and the anti-sera were collected. ELISA results showed that the mutant protein preserved its immunogenicity, particularly it could improve the production of antibodies to other epitopes in addition to the N terminal epitope of Tat protein, which might provide some valuable information for the study of Tat functions as well as for development of potential novel HIV Tat vaccine.
Animals ; Female ; Gene Products, tat ; biosynthesis ; genetics ; immunology ; HIV-1 ; genetics ; immunology ; Humans ; Mice ; Mice, Inbred BALB C ; Mutant Proteins ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVE: To synthesize the specific CagA gene segment of the gastric cancer idiotype Helicobacter pylori (H. pylori), establish the prokaryotic expression system and identify the antigenicity sequence of recombination signals. METHODS: We selected the CagA fragment which was related to gastric cancer in our earlier research. The CagA gene segment was optimized and synthesized. The synthesized CagA gene was cut from the pUC57-CagA plasmid and then was carried by expression vector pET32a to be transformed into the host bacterium BL21 (DE3). The positively cloned pET32a-CagA was selected by receptivity of aniline and colony PCR. The host bacterium with pET32a-CagA was induced by IPTG to express fusion protein. The expression of CagA protein was analyzed by SDS-PAGE gel electrophoresis and the antigenicity of fusion protein was examined by Western blot. RESULTS: CagA gene segment was designed and synthesized. The sequence of synthesis CagA gene segment was the same as the one designed before (AF289435). We successfully constructed the plasmid of prokaryotic expression of the pET32a-CagA. Homology of the target CagA proteinum was 100%, the same as AAG09884. The host bacterium BL21 (DE3) containing pET32a-CagA could express CagA fusion protein after the IPTG induction. SDS-PAGE gel electrophoresis showed that the molecular weight of fusion protein was the same as expected (45 kD). Western blot showed that the fusion protein could be combined with the antibody of the whole bacterium of anti-H. pylori. CONCLUSION The synthesized CagA fusion protein from the prokaryotic expression system has antigenicity. We hope to set the foundation for selecting the strain in H. pylori correlated to gastric cancer and corresponding therapy in clinical practice.
Amino Acid Sequence ; Antigens, Bacterial ; genetics ; immunology ; Bacterial Proteins ; genetics ; immunology ; Base Sequence ; Genetic Vectors ; Helicobacter pylori ; genetics ; immunology ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 7 (Nsp7) plays an important role in the induction of host humoral immune response and could serve as an ideal antigen for serological genotyping assay for PRRSV based on the significant difference in immunoreactivities of North American (NA) and European (EU) PRRSV Nsp7. In this study, Nsp7 of NA and EU PRRSVwas separately expressed and purified using prokaryotic expression system. The purified recombinant Nsp7 proteins reacted with serum antibodies against corresponding genotype PRRSV in Western blotting. However, nonspecific reaction of whole recombinant Nsp7 with antibodies against another genotype PRRSV was observed, indicating that whole NA PRRSV Nsp7 and EU PRRSV Nsp7 have similar antigenic epitopes and recombinant proteins could not be used for genotyping of antibodies against PRRSV. Based on the analysis of similar antigenic epitopes at the hydrophilic region of NA PRRSV Nsp7 and EU PRRSV Nsp7 by bioinformatics assessment, partial Nsp7 gene region deleted sequences encoding similar antigenic epitopes was constructed by fusion PCR. The recombinant truncated Nsp7 (NA-deltaNsp7 and EU-deltaNsp7, about 43 kDa) was expressed and the molecular weight was about 43 kDa. The results of Western blotting showed that NA-deltaNSP7 and EU-deltaNSP7 could be specifically recognized by positive serum to NA or EU PRRSV individually and nonspecific reaction was eliminated. This study provided a basis for further development of serological genotyping assay for North American and European genotype PRRSV infection.
Animals ; Genotype ; Porcine respiratory and reproductive syndrome virus ; classification ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; immunology ; Swine ; Viral Nonstructural Proteins ; biosynthesis ; immunology