The potential molecular effects of bursal septpeptide II on immune induction and antitumor activity

The bursa of Fabricius (BF) is the acknowledged central humoral immune organ in birds. Bursal septpeptide II (BSP-II) is an immunomodulatory bioactive peptide isolated from BF. To understand the effects of BSP-II on immune induction, gene expression profiles of hybridoma cells treated with BSP-II were evaluated. Pathway analysis showed that regulated genes were involved in cytokine-cytokine receptor interactions, T cell receptor signaling pathway, and pathway in cancer. It was observed that BSP-II reduced tumor cells proliferation and stimulated p53 expression. These results indicate potential mechanisms underlying the effects of the humoral immune system on immune induction, including antitumor activities. Our study has provided a novel insight into immunotherapeutic strategies for treating human tumors.

Influence of epitope A modification and N-linked glycosylated site mutation of PRRSV NJ-a strain ORF5 gene on the ability to induce neutralizing antibodies and T cell proliferation response / 生物工程学报

To enhance the DNA immunogencity of PRRSV ORF5 gene, CpG sequence and the universal helper T cell antigen epitope (PADRE) sequence were inserted between the decoy epitope and the neutralizing epitope. At the same time, site-mutations were introduced at N33 and N51 to diminish the coverage effect to epitope B from the polysaccharides. Subsequently, the modified ORF5 gene (MORF5) and PRRSV ORF6 gene were cloned into the eukaryotic expression vector pcDNA3.0 under the control of two CMV promoters, respectively. With indirect immunofluorescence assay and Western-blot the expression in vitro of the two genes was confirmed, then six-week-old Balb/C mouse were immunized with the modified expression plasmid pcDNA-M5A-6A. The non-modified expression plasmid pcDNA-5A-6A, the blank eukaryotic expression plasmid pcDNA3.0, living attenuated vaccine and inactivated vaccine were used as controls. The PRRSV specific neutralizing antibodies and the T cell proliferation response were elevated with virus neutralization assay and MTf method. Results indicate that the modified plasmid pcDNA-M5A-6A can elicit not only higher titer of neutralizing antibodies in a rapid time, but also more vigorous T cell proliferation response compared with the non-modified plasmid pcDNA-5A-6A and commercial vaccines, indicating that DNA vaccine pcDNA-M5A-6A maybe a promising candidate for PRRS prevention.

Secreted expression of the combinant antimicrobial peptide PL in Pichia pastoris and its antibacterial activity in vitro / 生物工程学报

In order to obtain a high activity antibacterial peptide, An expression vector pPICZalphaA-pl is constructed with a tandem of four antimicrobial peptides in the same direction,which includes Protegrin-1 (PG-1), Scorpion Defensin (SD), Metalnikowin-2A and Sheep Myeloid Antibacterial Peptide (SMAP-29) (serial number in GenBank are AAB27599, AAAB27538, P80409 and P49928 respectively). At the same time the expression vector pPICZalphaA-sd which express Scorpion Defensin was contructed. The expression vectors of pPICZalphaA-pl and pPICZalphaA-sd were linearized and transformed into the yeast host strain X-33 respectively. Under the control of the promoter AOX1 (alcohol oxidase1), the peptides PL and SD were secreted expressed. Their heat-stable property, acid-stable property and MIC were detected in vitro. The results suggest the peptides PL and SD have good heat-stable and acid-stable properties, and the combinant PL peptide showes higher antibacterial activity against several Gram-positive bacteria (G+) and Gram-negative bacteria (G-) than the peptide SD, especially against Escherichia coli. The antibacterial activity of combinant antimicrobial peptide PL shows its far exploiting perspective.

Fusion expression of O type foot-and-mouth diseases virus VP1 gene and HSP70 gene and induction of immune responses in mice / 生物工程学报

Vp1 gene of O type foot-and-mouth diseases virus and M. tuberculosis HSP70 were expressed in methylotrophic yeast Pichia pastoris expression system. The results of cellular immune responses and humoral immune response were examined after BALB/c mice were immunized with fusion protein expressed in methylotrophic yeast Pichia pastoris. The genes was cloned into the vector pPICZalpha-A by routine molecular technique. The plasmid fusion (pPICZalphaA-vp1-HSP70) was created that HSP70 located downstream of VP1 gene of O type foot-and-mouth disease virus. Vp1 was expressed by fusing to the amino terminus of M. tuberculosis hsp70 in yeast Pichia pastoris. The recombined fusion plasmid was transformed into methylotrophic yeast Pichia pastoris X-33 by electrophoration. The recombinant transformants were selected by Zeocin and induced by the addition of methanol every 24h. The expressived product analyzed by SDS-PAGE and Western blotting. The result indicated that the fusion protein(vp1-HSP70) has specific antigenicity. Mice were inoculated transcutaneous three times at a two-weeks interval with fusion protein, PBS and conventional inactivated vaccines. To evaluate the prophylaxtic efficacy of fusion protein, Titers of antibodies was detected by ELISA and proliferation of lymphocytes were determined by MTT. The results indicated that fusion protein could elicit specific humoral immune and cellular immune responses. Compared with conventional inactivated vaccines, fusion protein elicited slightly lower FMDV antibody level but stronger T cell proliferation.

Construction of TK Gene-deleted PRV SH StrainContaining a Single LoxP Site / 中国生物工程杂志

Pseudorabies virus (PRV) is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and economic losses worldwide. Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. A recombinant PRV which contain a loxP site in TK locus by using Cre/LoxP recombinant system was construsted. A pair of primers were synthesized according to the pEGFP-C1 sequence published on GenBank, and were used to amplify the EGFP gene expression cassette with two loxP sites flanking each side. This target gene was cloned into pSKLR, the resulting transfer vector pSKLR-GFP-loxP was then cotransfected into 293T cells with PRV SH strain genomic DNA. The recombinant virus rPRV1 was selected and purified in TK-143 cells by choosing fluorescent expressing plaques. Cre expression vector pOG231 was cotransfected into 293T cells with rPRV1 genomic DNA. The second recombinant virus rPRV2 was obtained, which contains only one loxP site in TK locus. Sequencing results of rPRV2 TK gene indicated that 34bp loxP site was inserted into rPRV2 genome and there were 270bp deletion in TK gene. PCR amplifying different generations of rPRV2 TK gene showed that the mutant was stable when passages in RK-13 cells. TCID_ 50 assay indicated that rPRV2 grows well on RK-13 cells. The LD_ 50 test results on BALB/C mice suggested that the virulence of rPRV2 was reduced. As a conclusion, the report gene GFP expression cassette was removed successfully from rPRV1 genome and only one LoxP site was leaved in rPRV2 genome by using Cre/LoxP recombinant system.

Gene modification and high prokaryotic expression of porcine interferon alpha-1 / 生物工程学报

There are many E. coli rare codons in the gene of porcine interferon alpha-1. In order to obtain high expression of poIFN-alpha1 in E. coli, the cDNA encoded poIFN-alpha1 mature protein was synthesized using biased codons of E. coli without changing the original amino acid sequence and the terminator was changed as TAA. At the same time, Adenine and Thymine were used to the largest extent near the 5' terminus of poIFN-alpha1 mature protein gene. The synthesized gene was inserted into the Eco RI and Sal I site of the expression vector pRLC resulting pRLC-poIFN-alpha1. The poIFN-alpha1 is highly expressed in E. coli DH5alpha when the induction was carried out at 42 degrees C . The expressed poIFN-alpha1 account for 24.5% of the total cellular proteins and existed as inclusion body. The poIFN-alpha1 inclusion body was dissolved in 6mol/L guanidine chloride contained DTT and subsequently the denatured poIFN-alpha1 was re-natured by dilution in refolding buffer containing GSH and GSSH. In the present study it was found that the denatured poIFN-alpha1 was most efficiently re-natured in refolding buffer containing 1 mol/L guanidine chloride. In order to obtain pure protein, the concentrated re-natured poIFN-alpha1 was purified by Sephacryl S-200 chromatography. As a result, the purified poIFN-alpha1 is verified to be of high cytokine activity by inhibiting the cytopathic effect of vesicular stomatitis virus in MDBK cells, which is about 6.4 x 10(6) u/mg. This study paved the way for large-scale production of recombinant poIFN-alpha1 and its usage in virus disease control of pigs.