Construction of srtA-deletion mutant of Streptococcus mutans by an in-frame deletion system / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To construct srtA-gene deletion mutant of Streptococcus mutans (S. mutans) UA159 with IFDC2 cassette through overlapping polymerase chain reaction (PCR) and allelic homologous recombination.</p><p><b>METHODS</b>First, the upstream and downstream fragments surrounding the srtA and IFDC2 cassette were PCR amplified and ligated through overlapping PCR. The resulting amplicon was transformed into UA159, and positive transformants were selected on BHI plates containing erythromycin. Second, upstream and downstream fragments of srtA with overlap regions were generated by PCR and were overlapped to create upΔ-down amplicon. Then, the upΔ-down amplicon was transformed into the aforementioned positive transformants and selected on BHI plates containing p-Cl-Phe.</p><p><b>RESULTS</b>The PCR analysis and DNA sequencing results indicated that the coding region of the srtA was completely deleted, and the upstream and downstream regions flanking the srtA were ligated seamlessly.</p><p><b>CONCLUSIONS</b>The markerless srtA-deletion mutant of S. mutans was constructed successfully, which laid a foundation for further study of its biological function and influence on the biofilm formation of S. mutans.</p>

Construction and characterization of a bovine herpesvirus 5 mutant with a deletion of the gI, gE and US9 genes

Bovine herpesvirus 5 (BoHV-5) is a important cause of viral encephalitis in cattle in South America. Within the framework of developing a differential vaccine against BoHV-5, a deletion mutant was constructed based on a Brazilian BoHV-5 isolate. The target of the deletions were genes that code proteins implicated in the neurovirulence of BoHV-5, the glycoprotein I (gI), glycoprotein E (gE) and membrane protein US9. To construct the deletion mutant of BoHV-5, the flanking regions of all three genes were cloned in a prokaryotic plasmid. This deletion fragment was co-transfected with the viral DNA into bovine cells. Identification of deletion mutants was performed by immunostaining with an anti-gE monoclonal antibody. One of the gE negative viral populations found was purified, amplified and further examined by restriction endonuclesase analysis of its genomic DNA. The plaque sizes and penetration kinetics of the deletion mutant and wild type viruses were compared. The plaque sizes of the deletion mutant were significantly smaller than those of the parental strain (p <= 0.05), but no statistical differences were observed in penetration kinetics. The results indicate that the gI/gE/US9 deletion mutant of BoHV-5 may have a reduced virulence in the host and is still viable enough to be a good candidate for the development of a BoHV-5 vaccine.

O herpesvírus bovino 5 (BoHV-5) é uma causa importante de encefalite viral em bovinos na América do Sul. Buscando o desenvolvimento de uma vacina diferencial contra o BoHV-5, um mutante deletado foi construído com base em um isolado brasileiro deste vírus. O alvo das deleções foram genes que codificam proteínas implicadas na neurovirulência do BoHV-5, a glicoproteína I (gI), a glicoproteína E (gE) e a proteína de membrana US9. Para construir o mutante deletado de BoHV-5, as regiões flanqueadoras dos três genes foram clonadas em um plasmídeo procarioto. Este fragmento de deleção foi co-transfectado com o DNA viral em células de bovinos. A identificação dos mutantes deletados foi feita por meio da técnica de imunoperoxidase com um anticorpo monoclonal anti-gE. Uma das populacões virais gE negativas encontradas foi purificada, amplificada e seu genoma foi examinado por análise de restrição enzimática. Os tamanhos de placas virais e taxas de penetração do vírus mutante foram determinados e comparados com os do vírus selvagem. As placas virais do vírus mutante deletado foram significativamente menores do que as do vírus selvagem (p <= 0,05), mas não foram encontradas diferenças significativas quando comparadas as taxas de penetração dos dois vírus. Estes resultados indicam que o vírus mutante deletado gI/gE/US9 de BoHV-5 pode apresentar virulência reduzida e é viável o suficiente para ser um bom candidato para o desenvolvimento de uma vacina contra o BoHV-5.

Construction of a middle fragment-deleted class Ⅱ molecule transactivator mutant by modified OE-PCR technique / 第二军医大学学报

Objective:To develop a simple and efficient method for constructing a middle fragment-deleted mutant of MHC class Ⅱ molecule transactivator(CⅡTA)mutant with the 109~(th)to 226~(th) amino acid codons deleted.Methods: Two gene fragments at each end of the deleted CⅡTA gene were obtained by OE-PCR method and were mixed together for 8 PCR cycles without primers to achieve effective overlapping,then 2 primers was added for amplification of the desired fragments.The amplification products were subsequently cloned into eukaryotic vector pIRES for identification.Results: A mutant of CⅡTA with the 109~(th)to 226~(th) amino acids deleted was successfully constructed.Conclusion: This modified OE-PCR technique overcomes some shortcomings of traditional method and is very suitable for constructing mutants with middle fragment deletion,making it worth to be popularized.

Deletion of the Importin-alpha Gene in the Breast Cancer Cell

BACKGROUND: BRCA1 (breast-cancer gene 1) is a tumor suppressor gene that accounts for nearly all families of both early onset breast and ovarian cancer and about 45% of families with breast cancer only. Sporadic nonhereditary breast cancer is recognized as the most common form of this malignancy. However, presence of germ-line mutations in the BRCA1 gene of these tumors is an infrequent event. The BRCA1 protein includes a ring domain and an acidic domain, both of which are characteristics of certain transcription factors, as well as two putative nuclear localization signals (NLS) that interact with importin-alpha. The normal BRCA1 protein is located in the nucleus of most breast-cell types whereas the BRCA1 protein of breast cancer cells is aberrantly localized in the cytoplasm. This mislocation of the BRCA1 protein in breast cancer cells may be due to defects in the NLS receptor-mediated pathway for the nuclear import of the BRCA1 gene product. Identification of importin-alpha mutations as a cellular protein responsible for the nuclear import of BRCA1 in breast-cancer cell lines and primary breast cancers is the focus of this investigation. METHODS: A series of 15 surgical samples of breast cancer and 3 samples of breast-cancer cell lines (Hs578T, ZR75-1, MCF-7) was assayed for the presence of the deletion mutant in importin-alpha by using both RT-PCR amplification of importin-alpha transcripts and sequencing analysis. RESULTS: Three of the 15 primary breast cancers and 1 of the 3 breast-cancer cell lines showing deletions in importin-alpha transcripts produced two different truncated transcripts. 1208 bp deletions were observed in transcripts from breast cancer (T-1, T-3) and ZR75-1, which is specified by the nucleotide 251-1458 of the transcript. Another transcript encoded by primary breast cancer (T-2) included a 1312 bp deletion in the nucleotide 61-1372 of the transcript. CONCLUSIONS: The deletions eliminated part of the importin-alpha transcript segment encoding the putative NLS-binding domain but not the importin-beta binding domain, suggesting that these deletion mutants could not bind to NLS of the BRCA1 protein. These results suggest that the composite effects of mislocationof the BRCA1 protein by deletion of the NLS-binding domain in importin-alpha may contribute to tumorigenesis in sporadic breast cancer.

RESEARCH ADVANCES ON PSEUDORABIES NEW-TYPE VACCINES / 微生物学通报

Pseudorabies is an important infectious disease for many kinds of livestock and wild animals, and causes important economics losses for pig industry. Many kinds of vaccines including attenuated live viruses or inactivated are widely used for vaccination of pigs and other animals. In the present review, research advances on pseudorabies new-type vaccines such as subunit vaccine, DNA vaccine, recombination vaccine, deletion-mutant vaccine is presented and point out the further development of the vaccine.

SITE-DIRECTED MUTAGENESIS OF TSR1 GENE IN YEAST YARROWIA LIPOLYTICA / 微生物学通报

The TSR1(thermosensitive rescued)gene associated with the secretion of proteins of the yeast Yarrowia lipolytica was site-directed mutagenesis,restricted and spliced to obtain a series of deletion mutants.These mutants would be useful for studing the function of different domains of TSR1 gene in yeast Yarrowia lipolytica

A preliminary study on targeting antigenic epitopes of monoclonal antibody against human TNF-? / 中国免疫学杂志

Objective:To locate the region of antigenic epitopes recognized by monoclonal antibody(Z8) against human tumor necrosis factor ?(hTNF ?).Methods:Several recombinant fusion expression vectors were constructed which contained hTNF ? mutant genes deleted different parts respectively. After induction with IPTG, the fusion proteins were expressed and analyzed by SDS PAGE and Western blot.Results:Z8 specially recognized the fusion products which contained amino acids from 92 to 157 of hTNF ?, but not GST and its fusion protein with amino acids from 1 to 91 of hTNF ?.Conclusion:The antigenic epitopes recognized by Z8 were located in the sequence region 92 157 of hTNF ?.