Cloning and Purification of Per a 4, a Gene Encoding a Periplaneta Americana Allergen, and Preparation of Its Monoclonal Antibodies.

OBJECTIVE: To clone, express, purify the Per a 4 gene encoding an allergen of Periplaneta Americana and prepare monoclonal antibodies against the recombinant allergen. METHODS: The total RNA was extracted from P. Americana, and the target gene was amplified by RT-PCR and cloned into pMD18-T vector. After being confirmed by nucleotide sequencing, the gene was then inserted into pGEX-3X to construct the express vector pGEX-3X-Per a 4. Further, the pGEX-3X-Per a 4 was transformed into E. coli BL21 (DE3), and induced for expression by IPTG. By affinity chromatography, the recombinant allergen was purified and identified by SDS-PAGE and Western blotting. The BALB/c mouse was immunized with the recombinant allergen to prepare the specific monoclonal antibodies, which was then identified by co-immunoprecipitatin and western blotting. RESULTS: The full-length cDNA encoding Per a 4 of P. Americana was obtained with 552 bp in length, which had 99.4% similarity with the reference sequence (GenBank AY792948). The constructed vector pGEX-3X-Per a 4 was transformed in E. coli BL21 (DE3), expressed with the induction of IPTG. By SDS-PAGE, a band of about 49 KD was present. Further, the western-blotting showed that the prepared monoclonal antibodies can bind the serum antibodies in patients allergic to P. Americana. CONCLUSIONS: Both the recombinant allergen Per a 4 and its monoclonal antibodies were obtained.

Analysis of chromosome in 1 324 patients with oligozoospermia or azoosperm / 北京大学学报(医学版)

OBJECTIVE@#To explore the incidience of chromosome abnormality of the patients with oligozoospermia or azoospermia and male infertility, to discuss the relationship between the quantitative and structural abnormality of chromosome and to lay the foundation for the clinical diagnosis and consultation.@*METHODS@#A retrospective analysis was conducted from January 1, 2015 to May 1, 2016, in the Center for Reproduction Medicine, the Second Hospital of Jilin University, with male reproductive abnormalities history excluded. In the study, 1 324 cases were included with 448 cases of azoospermia and 876 cases of oligozoospermia. All the patients through ultrasound examination, color Doppler ultrasonography, the seminal plasma Zn determination, their hormone level determination, chromosome karyotype (the perinatal blood samples were obtained from the 1 324 patients with oligozoospermia or azoospermia for lymphocyte culture, then chromosomal specimens were prepared, G-banding analyses combined with clinical data were used to statistically analyze the incidence of chromosomal abnormality), Y chromosome azoospermia factor [PCR technique was used to detect SY157 locus, SY254 locus, and SY255 locus in male Y chromosome azoospermia factor (AZF) gene of the patients with oligozoospermia or azoospermia]. The relationship between chromosome abnormalities and oligozoospermia or azoospermia were analyzed.@*RESULTS@#Among the 876 cases of oligospermia patients, 78 cases were chromosome number abnormality and chromosomal structural abnormality, the abnormal number of sex chromosomes in 22 cases, and sex chromosomes and chromosome structural abnormalities in 56 cases; in the 448 cases of azoospermia patients, 91 cases were chromosomal structural abnormality and chromosome number abnormality, of them, 78 cases were of abnormal number of sex chromosomes, and 13 cases were of abnormal structure. In addition, 137 cases were of chromosome polymorphism in all the 1 324 patients, The incidence of Y chromosome abnormality in azoospermatism was higher than that of the 43 patients with Y chromosome AZF microdeletion. In addition, the asthenospermia and recurrent spontaneous abortion were closely related to Y chromosome abnormality and the chromosome translocations and inversions.@*CONCLUSION@#Oligozoospermia and azoospermia patients with abnormal chromosome karyotype have high incidence rate, and chromosome karyotype analyses were carried out on it, which is conducive to clinical diagnosis for the patients with abnormal chromosome karyotype. There is a close relationship between male infertility and abnormal karyotype. It is conducive to clinical diagnosis for the patients with infertility through chromosome karyotye analysis, which also provides evidence for genetic counseling.

UPLC-Q-TOF/MS for rapid analysis of chemical constituents in Sanhuang tablets / 中国中药杂志

The qualitative analysis method of ultra performance liquid chromatography tandem quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS/MS) was established for the chemical constituents in Sanhuang tablets. Waters ACQUITY BEH C₁₈ (2.1 mm×100 mm, 1.7 μm) column was used with 0.1% formic acid solution (A)-0.1% formic acid acetonitrile (B) as the mobile phase for gradient elution. The flow rate was 0.2 mL•min⁻¹; the sample volume was 1 μL and the column temperature was 30 ℃. The high-resolution quadrupole time-flight mass spectrometry was used as detector with electrospray ion source in both positive and negative models, and the dry gas temperature was 325 ℃. Based on the analysis of mass spectrometry and literature reports, 38 compounds were confirmed, including 1 alkaloid, 1 dianthrone compound, 6 tannins, 7 anthraquinone glycosides, 6 anthraquinones and 17 flavonoids. Liquid chromatography-mass spectrometry method is simple, reliable and rapid to identify the chemical compositions of Sanhuang tablets, and it is helpful to reveal its chemical constituents and pharmacodynamic substances.

Practice and thinking on teaching of medical genetics in medical students / 中华医学教育探索杂志

Medical genetics is one of the important basic courses in medical education. The teaching reform in course content, teaching method and experimental teaching was carried out to arouse their enthusiasm in study, cultivate their capabilities of analyzing of medical practice problem.

Autophagy is involved in 6-OHDA-induced dopaminergic cell death / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the role of autophagy in the death of dopaminergic neurons induced by 6-hydroxydopamine (6-OHDA).</p><p><b>METHODS</b>Rat models of Parkinson disease (PD) were established by stereotaxic administration of 6-OHDA (8 μg) into the unilateral substantia nigra par compact (SNpc). Autophagosomes in the SNpc were observed with transmission electron microscopy (TEM), and the expression of autophagy-related protein LC3 was determined with immunofluorescence (IF) assay.</p><p><b>RESULTS</b>Under TEM, the autophagosomes were found in the ipsilateral SNpc 6-24 h after 6-OHDA injection, which suggested the activation of autophagy. IF assay showed significantly increased LC3 expression in 6-OHDA-damaged TH-positive neurons as compared to the control group.</p><p><b>CONCLUSIONS</b>The increase of autophagosomes and activation of autophagy may play a role in dopaminergic neuron death induced by 6-OHDA.</p>

Expression of porcine beta-defensin 1 gene in Pichia pastoris / 生物工程学报

PBD-1 is an antibacterial peptide that plays an important role in defence system of porcine. To produce PBD-1 with bioactivity in Pichia pastoris, according to published amino acid sequence of porcine beta-defensin 1(PBD-1) and the partiality codon of yeast, the PBD-1 gene was synthesized by PCR and cloned into pPIC9K to construct the recombinant expression vector pPIC9K-PBD-1, the obtained recombinant plasmid was linearized by Sal I, and then transformed into SMD1168 by electroporation. Under the control of the promoter AOX1, an approximately 4.5 kD PBD-1 peptide was expressed. Antibacterial activity assay shows that the PBD-1 has the antibacterial activity on Staphylococcus aureus. This is the first secreted expression of porcine beta-defensin 1 gene in Pichia pastoris.

Expression and Antigenic Analysis of the Recombinant Epitope of Herpes Simplex Virus Type 2 Glycoprotein G / 中国生物工程杂志

A fragment containing amino acid residues 561～578 of HSV-2 glycoprotein G(gG2) was obtained by PCR assembling technique,and doubly cloned into vector pET-KDO.The recombinant plasmid was transformed to BL21(DE3)plysS.Fusion protein,of molecular weight about 39kDa was highly expressed by induction of IPTG.Western blot result showed the fusion protein had good antigenicity.After putification and digestion,the purity reached 95%.The digested purified protein was analysed by ELISA and showed good sensitivity and specificity.The recombinant protein should be useful for type-specific serodiagnosis of HSV-2.

High Efficiency Expression of the Gene From Herpes Simplex Virus Type 1(HSV-1)Glycoprotein D / 中国生物工程杂志

Objective:To obtain the high expression of Herpes Simplex Virus Type 1(HSV-1)Glycoprotein D gene. Methods:The Herpes Simplex Virus Type 1(HSV-1)Glycoprotein D(gD1) gene fragment containing dominant antigen epitopes confirmed by computer analysis was cloned by PCR technical and inserted into plasmid vector pTrxA. Then the recombinant plasmid was transformed into Rosetta. The expressed product was analyze by SDS-PAGE. Results:930 bp gene fragment was amplified by PCR as anticipated. Nucleotide sequencing showed a 100 % homology with that of the published sequence in GenBank. The molecular weight of the expressed protein was about 48kDa, Western blotting indicated that the antigenicity of the protein was good. Conclusion:The plasmid pTrxA-gd1 was constructed and a high efficiency expression of the gd1 gene from Herpes Simplex Virus Type 1(HSV-1)strain was made. The expressed product shows a good antigenicity.

Medical images edge detection based on mathematical morphology.

Medical images edge detection is an important work for object recognition of the human organs and it is an important pre-processing step in medical image segmentation and 3D reconstruction. Conventionally, edge is detected according to some early brought forward algorithms such as gradient-based algorithm and template-based algorithm, but they are not so good for noise medical image edge detection. In this paper, basic mathematical morphological theory and operations are introduced at first, and then a novel mathematical morphological edge detection algorithm is proposed to detect the edge of lungs CT image with salt-and-pepper noise. The experimental results show that the proposed algorithm is more efficient for medical image denoising and edge detection than the usually used template-based edge detection algorithms and general morphological edge detection algorithms.

Development of a Portable Blood Sugar Apparatus and GOD Enzyme Strip.

A pocket blood sugar apparatus tested by enzyme electrode, which was designed using carbon and silver plasma as conducting materials. Both the work and reference electrodes are applied to the parts of enzyme electrode. The glucose oxidase is taken as the medium of blood sugar measuring. And the range of measuring glucose is about 50mg/dL - 500mgl/dL. It has better linear for the results and fit coefficient arrives at 0.985. Its sensitivity of measurement is higher than current glucose biosensor obviously. A single chip microcomputer, AD mu C812, is used for central control processor of the instrument parts. It measures the output of microampere level currency, which is conduced by blood sugar reacting with the glucose oxidase on the enzyme electrode. And at the same time, the microampere level currency is amplified, processed. Then the results are displayed on LCD. The apparatus are better for measuring blood sugar, and the results are consistent with what the large biochemical instruments get.

A case-control study on the polymorphisms of methylenetetrahydrofolate reductases, drinking interaction and susceptibility in colorectal cancer / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between methylenetetrahydrofolate reductases (MTHFR) polymorphisms and colorectal cancer susceptibility.</p><p><b>METHODS</b>A case-control study of 126 patients and 343 healthy controls was conducted to investigate the roles of MTHFR C677T and A1298C polymorphisms in colorectal cancer development. Genotypes of C677T and A1298C polymorphisms were analyzed by polymerase chain resction-restriction fragment length polymorphism (PCR-RFLP) methods.</p><p><b>RESULTS</b>The frequencies of MTHFR 677T and 1298C allele were 39.7% and 17.1%, respectively. After adjustment for age and sex, the MTHFR 1298C alleles seemed to have reduced association on the risk of colorectal cancer comparing to wild types. Among those with 677T and 1298A alleles, a decreased risk of colorectal cancer was observed: a 4-fold decrease in colorectal cancer risk (OR = 0.552, 95% CI: 0.265 - 1.150) in those with 677T and 1298C alleles. Men who were ex-drinkers and with MTHFR 1298C allele had a 2-fold increase in risk of colorectal cancer (OR = 3.307, 95% CI: 0.521 - 17.698) while no increased risk was seen among those current-drinkers.</p><p><b>CONCLUSIONS</b>This study suggested that certain MTHFR C677T and A1298C might be associated with the risk of colorectal cancer development. The interaction between MTHFR 1298AC polymorphisms and ex-drinking might also serve as a risk factor of colorectal cancer.</p>

Expression and Identification of LfcinB Gene in Pichia pastoris / 微生物学通报

In present study,bovine Lactoferricin was first secretly expressed in Pichia pastors yeast expression system.The synthesized LfcinB gene fragment was cloned into expression vector pPIC9K,and then obtained recombinant plasmid,designated as pPIC9K-LfcinB,was linearized and transformed into Pichia pastors strains SMD1168 by electroporation.The transformants were screened with Geneticin and multiply-copy colonies were harvested,in which LfcinB gene was verified to inserted into yeast chromosome stably.The positive recombinant Pichia strains were induced with methanol to express LfcinB in culture supernatant.It's expressive products has high activity of killing bacteria.We concluded that LfcinB gene was cloned and integrated into yeast chromosomes,and obtained expression peptide was tested to have high antibacterial activity.