Research progress of lncRNA, miRNA and circRNA in central nervous system diseases / 医学研究生学报

Regulatory noncoding RNA (ncRNA), mainly including long noncoding RNA (lncRNA), microRNA (miRNA) and circular RNA (circRNA), is one of the research hotspots at home and abroad in recent years. Numerous studies have shown that LncRNA, miRNA and circRNA are significantly differentially expressed and play an important role in central nervous system diseases, which may become the potential targets for novel diagnostic markers and for the treatment of diseases. In this paper, the differential expression, possible mechanism and the latest research progress of lncRNA, miRNA and circRNA in central nervous system diseases are reviewed in order to provide reference for further research.

Codon optimization of recombinant staphylococcal enterotoxin O enhances the expression level in Escherichia coli / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To enhance the expression level of staphylococcal enterotoxin O (SEO) by optimization of rare codons.</p><p><b>METHODS</b>The gene of mature SEO (His-tag included) was cloned to pET28a, and 15 rare codons on the gene were optimized by PCR technology. These recombinant plasmids then were transformed into E.coli BL21(DE3), respectively. After IPTG induced, the expression levels of those mutants were analyzed by SDS-PAGE. The proteins were purified and their bioactivities were determined.</p><p><b>RESULT</b>After the optimization of rare codons, the expression levels were increased from 7.49% to 19.8% in total cell proteins. The optimized SEO had bioactivity to stimulate the proliferation of murine lymphocytes, which was equivalent to that of non-optimized SEO in vitro.</p><p><b>CONCLUSION</b>Optimization of rare codons can enhance the expression of SEO effectively.</p>

Relationship between rennin and angiotensinogen gene polymorphisms and cerebral infarction / 中国脑血管病杂志

Objective: To study the relationship between the renin (REN) gene G10631A, angiotensinogen (AGT) gene T704C, C521T mononucleotide polymorphisms and cerebral infarction. Methods: One hundred eighty patients with cerebral infarction and 130 healthy controls were recruited. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to detect the REN G10631 site, AGT T704 and C521 site genotype and allele. The differences of the genotype and allele frequencies in both groups were compared. Logistic regression was used to analyze the risk factors for cerebral infarction. The haplotype structure of the population was analyzed in order to find cerebral infarction related polymorphism combination in this population. Results: Circled digit oneThe REN 10631 AA genotype frequency (31.7%) and the A allele frequency (49.4% ) in the cerebral infarction group were high-er than 10.0% and 30.3% in the healthy control group (P<0.05). Circled digit twoThe AGT 704 CC genotype frequency (63.3% ) and C allele frequency (79.7% ) in the cerebral infarction group were higher than 34.6% and 61.2% in the healthy control group (P<0.05). Circled digit threeThe AGT 521TT genotype frequency (21.7% ) and T allele frequency (27.8%) in the cerebral infarction group were higher than 6.9% and 11.9% in the healthy control group (P<0.05). Circled digit fourMultivariate Logistic regression analysis showed that REN 10631AA genetype, AGT 704CC genetype, and AGT52ITT genetype could increase the probability of cerebral infarction. The relative risk (OR) of the onset was 2.617, 2.699, and 3.362, respectively (P < 0.05); Circled digit fiveThe distribution frequency of the haplotype 521T-10631A-704C in the cerebral infarction group was higher than that in the healthy control group (P = 0.000). Conclusion: REN gene 10631AA genetype and A allele, ACT 704CC genetype and C allele, and AGT 521TT genetype and T allele may be the susceptible factors of cerebral infarction. Haplotype 521T-10631A-704C may be the genetic risk factors for the onset of cerebral infarction.

Preparation and identification of monoclonal antibodies to staphylococcal enterotoxin I / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare and identify monoclonal antibodies against staphylococcal enterotoxin I (SEI).</p><p><b>METHODS</b>Spleen cells obtained from mice immunized with the SEI protein were fused with the myeloma cells (SP2/0). Hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) and the stable monoclonal hybridomas were isolated by limiting dilution at least three times. The characters of purified monoclonal antibodies were identified by indirect ELISA and Western blotting.</p><p><b>RESULT</b>The monoclonal antibodies secreted by two hybridomas 8F7 and D8 belonged to IgG(2b) and IgG(1) subtypes. Both had high titer and specificity with no cross reaction to SEG, SEE and SEC.</p><p><b>CONCLUSION</b>The monoclonal antibodies against SEI has been successfully prepared and identified in this study.</p>

Safety and immunological effect of domestic split influenza virus vaccine / 中华预防医学杂志

<p><b>OBJECTIVE</b>To evaluate the safety and immunological effect of domestic split influenza virus vaccine.</p><p><b>METHODS</b>All 606 subjects were divided into three groups by under 6, 16-60 and above 60 years old. Each age group was divided as study group (n = 213), control group 1 (n = 195) and control group 2 (n= 198) by Table of Random Number, one domestic vaccine and two imported vaccines were respectively inoculated in three group people. The differences of clinical side effect rate, antibody positive rate, protective rate and geometric mean titer (GMT) of these three vaccines were compared by using the statistical software with statistical significance of P < 0.05.</p><p><b>RESULTS</b>The side effect rate of study group, control group 1 and control group 2 was 3.76% (8/213), 4.10% (8/195), and 3.54% (7/198), respectively without statistical significance(chi2 = 0.87, P =0.93). The positive seroconversion rates of H1N1, H3N2 and B in these three groups were respectively 89.2% (190/213), 63.4% (135/213), 86.4% (184/213), 88.7% (173/195), 61.5% (120/195), 87.2% (170/195), 87.9% (174/198), 61.6% (122/198) and 84.8% (168/198). There were no statistical significance in the total positive seroconversion rate of each antibody type (chi2(H1N1) = 0.94, P(H1N1) = 0.63; chi2(H3N2) = 0.94, P(H3N2) = 0.63; chi2(B) = 0.75, P(B) = 0.69). The average growth multiple of H1N1, H3N2 and B in these three groups were 10.7, 7.3, 8.4, 10.5, 6.3, 8.3, 10.2, 7.1, 8.8 times. There were no statistical significances in the GMT growth multiple of each antibody type (F(H1N1) = 0.35, P(H1N1) = 0.70; F(H3N2) = 2.22, P(H3N2) = 0.11; F(B) = 1.51, P(B) = 0.35). The antibody protective rates of H1N1, H3N2 and B were 100% (213/213), 70.0% (149/213), 95.3% (203/213), 100% (195/195), 66.7% (130/195), 97.9% (191/195), 99.5% (197/198), 66.2% (131/198), 96.5% (191/198) respectively. There was no statistical difference among the three vaccines (chi2(H1N1) = 2.04, P(H1N1) = 0.36; chi2(H3N2) = 0.74, P(H3N2) = 0.69; chi2(B) = 0.42, P(B) = 0.82).</p><p><b>CONCLUSION</b>The domestic influenza split vaccine might be suitable for colony vaccination for its having clinical safety and immunological effect.</p>

Peptide bond scission of staphylococcal enterotoxin C2 and related factors / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the limited digestion of recombinant staphylococcal enterotoxin C2 (SEC2-His)in different conditions.</p><p><b>METHODS</b>The purified recombinant SEC2-His was treated with different reagents and the cleavage of rSEC2 molecule was observed by SDS-PAGE.</p><p><b>RESULT</b>The cleavage occurred in positions Cys93-Cys110 of the disulfide loop. Complete auto-cleavage of recombinant SEC2 was observed in solution at 37degrees within 24 hrs, and that was accelerated under alkaline conditions. The auto-cleavage of the recombinant protein was inhibited in the presence of beta-ME (2%), PMSF (5-10 mmol/L), imidazole (1 mol/L) or crude E.coli lysate. Non-specific degradation of recombinant SEC2 was promoted with the increasing of the concentration of H(2)O(2).</p><p><b>CONCLUSION</b>The recombinant SEC2-His is broken down in special site of protein, which may be associated with the protein structure.</p>

Coexpression of PXRLBD with SRC88 and construction of equilibrium dialysis model of screening PXR ligands / 药学学报

The aim of this study was to obtain the soluble protein of human pregnane X receptor ligand binding domain (PXRLBD) through the coexpression of PXRLBD and 88 amino acids of steroid receptor coactivator-1 (SRC88) and apply the protein to constructing a new model of screening PXR ligands. Expression plasmid of pETDuet-1-SRC88-PXRLBD was constructed and transformed into Escherichia coli Rosetta (DE3) to coexpress PXRLBD and SRC88 via induction by IPTG at low temperature. Then an equilibrium dialysis model was constructed to study the interaction between PXRLBD and drugs including clotrimazole and dexamethasone, using HPLC as the analysis method. The results showed that the soluble protein of PXRLBD was obtained and the HPLC data indicated that clotrimazole bound to PXRLBD, while dexamethasone did not bind to PXRLBD, which indicated the successful establishment of a new method for studying the interaction between PXR and drugs. The new method may be useful in the screening of PXR ligands in vitro.

Preparation and application of antibody against staphylococcal enterotoxin C2 / 药学学报

The filtrate of Staphylococcus aureus culture has been used in an ampule form named as staphylococcal enterotoxin C injection for cancer therapy in clinic for ten years in China and proved to be effective. The active constituent of three kinds of injections is claimed to be staphylococcal enterotoxin C2 (SEC2), and the content of SEC2 is used as quality control. However, the correct content of SEC2 was not known and the relative amount of SEC2 was very low because of the complicated components of the filtrate. In this research, we established a proper ELISA system for the detection of SEC2 in staphylococcal enterotoxin C injection, which will improve the quality control of the injection. We produced and identified polyclonal and monoclonal antibodies of SEC2 and established BA-ELISA method based on the method of sandwich ELISA. It was found that the BA-ELISA method had good specificity, sensitivity and reproducibility, and being able to detect SEC2 at concentration from 2 to 20 ng x mL(-1), with an average CV value of 5.08%. The SEC2 content in staphylococcal enterotoxin C injection was calculated. There is some difference between the actual and labeled contents in the injections.

Alternatively spliced variants of constitutive androstane receptor in liver of mouse / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the existence of alternatively spliced variants of constitutive androstane receptor (CAR) in liver of mouse.</p><p><b>METHODS</b>The nucleotide from liver of mouse was purified and the CAR cDNA was amplified by PCR. The fragments of CAR cDNA were cloned to T vector and sequence analysis was performed.</p><p><b>RESULT</b>Various spliced variants of CAR in liver mouse were confirmed by DNA sequencing.</p><p><b>CONCLUSION</b>There are alternatively spliced variants in CAR, which are located in the ligand binding sequence of CAR.</p>

Construction, expression and characterization of recombinant fusion protein HSA-PTH (1-34) in Pichia pastoris / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To obtain recombinant fusion protein HSA (human serum albumin)-PTH(1-34) in Pichia pastoris.</p><p><b>METHODS</b>HSA and PTH(1-34) cDNA were obtained with PCR and the DNA segments were cloned into vector pPIC9 with linker. The linearized plasmids were transformed GS115 competent cells treated with LiCl, and mut+ transformants were screened on MD plate. With AOX promoter and alpha-MF signal sequences leading, fusion protein was expressed in GS115. PCR and SDS-PAGE were employed to confirm the integration and expression of HSA-PTH(1-34). The fusion protein was identified by Western blotting and classical adenylate cyclase assay.</p><p><b>RESULT</b>The PCR results showed that the gene of HSA-PTH(1-34) was integrated into GS115 genome. Western bolt approved the existence of two domains of HSA and PTH(1-34). The bioactivity assay in rabbit cortical membranes indicated that HSA-PTH (1-34) activated adenylate cyclase, but the activity was lower than that of the synthetic PTH(1-34).</p><p><b>CONCLUSION</b>Active fusion protein HSA-PTH (1-34) is successfully expressed in Pichia pastoris.</p>

Expression and bioactivity of the cloned staphylococcal enterotoxin O / 药学学报

This study is to clone the gene of staphylococcal enterotoxins O, obtain recombinant protein (rSEO) and investigate its activity on mice lymphocyte. Staphylococcus aureus O gene is cloned into GST gene fusion vector pGEX-4T-1. The resultant plasmid pGEX-4T-SEO was used to transform E. coLi BL21, where the GST-SEO fusion protein was expressed efficiently. Then SEO was purified by Glutathione Sepharose 4B affinity column and digested with thrombin. The bioactivity of SEO was analyzed by MTT assay on mice lymphocyte and tumor cells. The nucleotide sequence was confirmed to code for the protein correctly, and soluble SEO was expressed efficiently in E. coli BL21 with pGEX-4T-SEO. The protein purified by affinity chromatography resulted to be one single band by SDS-PAGE detection. The MTT assay of the purified rSEO demonstrated that its abilities of stimulating T cells and inhibiting the proliferation of K562, K562-ADM and B16 cells were equivalent to that of SEC in vitro. The expression plasmid pGEX-4T-SEO was constructed and the recombinant superantigen was expressed successfully, which may provide a foundation for the further research of the anticancer activity of SEO.

Expression and bioactivity analysis of staphylococcal enterotoxin C2 / 药学学报

<p><b>AIM</b>To clone the gene of staphylococcal enterotoxin C2 and express it in the form of a soluble fusion protein in E. coli. Then the activation of SEC2 on mice lymphocyte and its lethal effects on tumor cells were studied.</p><p><b>METHODS</b>Staphylococcus aureus SEC2 gene was cloned into GST gene fusion vector pGEX-4T-1. The resultant plasmid pGEX-4T-SEC2 was used to transform E. coli BL21, where the GST-SEC2 fusion protein was expressed efficiently. The rSEC2 protein was purified with Glutathione Sepharose 4B affinity column and digested with thrombin. The in vitro culture system was utilized to observe the activation of the SEC2 on mice lymphocyte and the lethal effects on tumor cells of the activated mice lymphocyte.</p><p><b>RESULTS</b>The proper gene of SEC2 was cloned and purified rSEC2 was obtained. The MTT results indicated that rSEC2 have strong ability to stimulate mice lymphocyte to proliferate with a dose-dependent manner. With the proliferation of mice splenic lymphocyte, rSEC2 has a strong lethal effect on tumor cells B16, K562 and K562-AD.</p><p><b>CONCLUSION</b>In this study, the gene of SEC2 was cloned and the rSEC2 protein was obtained, which had strong lethal effect on tumor cells B16, K562 and K562-AD.</p>