[Codon optimization and expression in Pichia pastoris of E2 gene of classical swine fever virus].

Codon bias was one of the important parameter which influence heterogenous gene expression, optimizing codon sequence could improve expression level of heterogenous gene. In the preview study, wildtype E2 gene was expressed poorly in Pichia pastoris, in order to improve the expression level of E2 gene in Pichia pastoris, the low usage codons of E2 gene were mutated into high usage codons in Pichia pastoris by directed-mutagenesis based on PCR. The result showed that, compared with the results reported in preview study, the expression level of E2 gene in Pichia pastoris was improved observably by substituting 24 low usage codons of E2 gene for the high usage synonymous codons.

[Expression of antigenic region of VP1 gene of swine vesicular disease in Escherichia coli].

The antigenic region of VP1 gene of swine vesicular disease virus was amplified by reverse transcription polymerase chain reaction (RT-PCR) and nested polymerase chain reaction (nPCR). After the amplified fragment was cloned into the expression vector pProEX-HTb. The insert position,the size and the reading frame of the insertion were identified by PCR, restriction digestion and sequence analysis of the recombinant plasmids.

[Expression of recombinant plasmid pcDNA3.1/P12X3C with multi-genes of foot-and-mouth disease virus in BHK-21 cells].

In order to obtain the gene P12X3C of Foot-and-Mouth Disease Virus (FMDV) that includes full length P1, 2A, 3C and a part of 2B, the site mutation strategy was used. After being digested by Kpn I and Xba I respectively, the gene P12X3C was cloned into the pcDNA3.1 (+) expression vector.

Immune response in guinea pigs vaccinated with DNA vaccine of foot-and-mouth disease virus O/China99.

In order to obtain the gene P12X3C of foot-and-mouth disease virus (FMDV O/China99) that includes full length P1, 2A, 3C and part of 2B and 3B, the site mutation strategy was used. The recombinant plasmid pcDNA3.1/P12X3C was transfected into BHK-21 cells.

The expression of SARS-CoV M gene in P. Pastoris and the diagnostic utility of the expression product.

High-level protein expression is an important means of obtaining large amounts of viral proteins to investigate further their biological properties. To express the membrane (M) protein of SARS-CoV at high-level in vitro, the M gene fragment was amplified and cloned it into the Pichia Pastoris expression vector pPICZalphaA. SDS-PAGE and Western blotting analysis of the induced products of recombinant yeast transformant indicated that successful high-level expression of M protein was achieved, and that the expression product was similar antigenically to the natural protein.

The effect of bovine IFN-alpha on the immune response in guinea pigs vaccinated with DNA vaccine of foot-and-mouth disease virus.

In this study, we constructed recombinant plasmid pcDNA3.1/P12X3C3D including P1, 2A, 3C, 3D and part of 2B gene of FMDV and pcDNA3.1/IFN containing the gene encoding bovine IFN-alpha.

Generation of an infectious cDNA clone of an FMDV strain isolated from swine.

A full-length cDNA clone of a foot-and-mouth disease virus (FMDV) isolated from swine was assembled in, the plasmid vector pBluescript II SK+ downstream of a T7 promoter. RNA synthesized in vitro using T7 polymerase lead to the production of infectious particles upon transfection of BHK-21 cells, as shown by cytopathic effects. The rescued virus was also found to be highly pathogenic for mice by intradermal injection producing a fatal disease indistinguishable from that of wild-type virus.

[Effects of lanthanum on alkaline phosphatase].

Objective: To investigate the effects of lanthanum on the activities of two kinds of alkaline phosphatase (AP)-rat liver AP and bovine intestine AP, and the preliminary mechanism involved.

Methods: A traditional colorimetric method was used to measure the activity of AP. The influence of La3+ on the intrinsic fluorescence of protein was studied by the method of fluorescence titration into the AP solution by LaCl3.

[Cloning of the major antigen region of E2 gene of hog cholera virus and expression in Escherichia coli].

The major antigen region of E2 gene of Hog Cholera Prevalent Strain (Guangxi Yuling Strain) and Chinese Hog Cholera Lapinised Virus (C-strain) derived from hog and rabbit spleen tissue, was amplified by reverse transcription polymerase chain reaction(RT-PCR) and the nested Polymerase Chain Reaction (nPCR). After the amplified fragments were cloned into the expression vector pPROEX-HTb, the recombinant plasmids pPROEX-GXYL and pPROEX-C were obtained. The insert position, the size and the reading frame were right by PCR, restriction digestion and the sequence analysis.

[Study on the expression of E2 gene of classical swine fever virus in Pichia pastoris and the immunological activity of its expression product].

E2 gene of classical swine fever virus (CSFV) was cloned into secretory pPIC9K Pichia pastoris expression vector. After being linearized by digestion, the vector was transformed into Pichia pastoris by electroporation to integrate with the genome, the transformants with high copies were screened by G418 and were induced to express with methonal. The results of SDS-PAGE and Western blot demonstrated that the supernatant of the induced P.

Relationship between bilirubin free radical and formation of pigment gallstone.

In this paper, we summarize the main progresses made in our group in the field of the mechanism of pigment gallstone formation. It was found that after treatment with free radicals, bilirubin (BR) was changed into free radical itself, and a semiquinone free radical and a superoxide free radical bound with metal were recognized, which was detected by ESR (electron spin resonance). By the means of NMR (nuclear magnetic resonance) and IR (Infra-red spectra), it was postulated that bilirubin polymerized through the reaction between the vinyl group and the hydroxyl group under the attack of free radicals.