The recombinant protein drugs have the advantages of high activity,high specificity and low toxicity,which have a very broad application prospect in the world.At present,the recombinant therapeutic proteins in vitro are produced from prokaryotic and eukaryotic expression system in order to achieve efficient expression.In view of the advantages such as perfect post-translational modification and protein folding,eukaryotic expression system has been widely used in the production of recombinant protein drugs.There are four commonly used eukaryotic expression systems,including yeast,insect cell,mammalian cell and plant cell expression systems.In this paper,the characteristics,applications and prospects of several common eukaryotic expression systems are reviewed,in view to providing a reference for the selection of suitable expression systems for recombinant protein drugs and promoting the research and development of new drugs.

A method of observation, examination and record was introduced in this paper, which was applicable for microorganism materials (example : bacteria, yeast moulds), animal and plant materials (example: animal tissue, flowers), bio-masromolecule (example: starch), industrial crystals, metallograph tests and general physics tests, through the hyphae, spores, spore sacs, expanding hyphae in fermentation and riboflavin crystals of E. ashbyii T30 (a riboflavin high-yield strain) had been observed.

BACKGROUND: Matrix attachment region (MAR), a DNA sequence, is still bound to the nuclear matrices after chromatindigested with restriction endonuclease, not only affects expression of endogenous gene, but also overcames transgenic silence andimproves transcription and expression of exogenous gene. OBJECTIVE: To investigate the influence of ?-interferon MAR of CHO cells on the transgenic expression of chloramphenicolacetyltransferase (CAT). DESIGN, TIME AND SETTING: The opening experiment was performed at the Department of Biochemistry and MolecularBiology, Molecular Institute, Xinxiang Medical College from October 2006 to April 2007. MATERIALS: CHO cell lines were obtained from China Center for Type Culture Collection. The pCATG vector of CAT and G418screening markers were constructed by this laboratory. METHODS: Human ?-interferon MAR by PCR was digested with SacI/KpnI and BamHI/SalI, and was inserted into pCATGvector, which was propagated in Escherichia coli JM109, then extracted and purified followed by enzyme digestion analysis. Vectorof CAT expression cassette and human ?-interferon MAR by the two sides was successfully constructed, and christened aspCAT-MAR. Two methods were compared between CHO cells of pCATG transformation and CHO cells of pCATG-MARtransformation. After G418 selecting, genome DNA of cell lines of G418 was extracted, then primers for PCR to amplify the CATtarget gene fragment was designed. MAIN OUTCOME MEASURES: The activity of CAT was analyzed by ELISA method. It was also tested to see if thepCATG-MAR was stably integrated into genomic DNA in the transfected cells. RESULTS: CHO cells of pCATG transformation was screened to have 16 strains of positive cell, and CHO cells of pCATG-MARtransformation was screened to have 17 strains of positive cell. Human ?-interferon MAR could increase the CAT gene expressionby 2.8 fold. The coefficient of variation of CHO cells of pCATG transformation was 2.065 0, and coefficient of variation of CHOcells of pCATG-MAR transformation was 0.813 1. Genome DNA of stable transformation cell lines was amplified by a fragment of437 bp. The results confirmed the pCAT-MAR vector was stably integrated into genomic DNA. CONCLUSION: Human ?-interferon MAR can increase transgenic expression in CHO cells and decrease the transgenicexpression variation in different transfected cells.

Objective To analyze the effect of GC-rich DNA fragments on the level of transgenic expression in Chinese hamster ovary (CHO) celts and its position effect.Methods The synthetic DNA fragment with GC-rich was cloned into the 5'or 3'or both 5'and 3'ends of expression cassette of expression vector.Three new expression vectors (pIRES-G1,pIRES-G2 and pIRES-G3) which was inserted with the GC-rich DNA fragments in different position were transfected CHO ceils,respectively,and then was observed under fluorescence microscope;the control vector was pIRES-EGFP.Stable transfected cell lines were screened under G418,and enhanced green fluorescent protein(EGFP) expression was analyzed by flow cytometry and the transgenic copy number was detected by quantitative real-time quantitative polymerase chain reaction (qRT-PCR).Results Three expression vectors with a GC-rich DNA fragments in different position were constructed successfully.The insertion of GC-rich DNA fragments at 3'end and both 5',3'ends of the box of expression vector could obviously improve the expression level of vector in CHO cells;and the expression level of the stably transfected CHO cells increased 1.39 fold and 1.32 fold compared to the control vector,respectively;the transgene copy number increased 1.32 fold and 1.24 fold compared with the control vector.While the insertion of GC-rich DNA fragments at 5'end of expression cassette had no obvious effect on the level of gene expression.Conclusion The role of DNA fragment with GC-rich in improving the transgenic expression of CHO cells is related to its position in the vector.The insertion of GC-rich DNA fragments at 3'end and both 5',3'ends of the box of expression vector can improve transgenic expression.

BACKGROUND:Matrix attachment region(MAR)are DNA elements that bound to the nuclear matrices after chromatin digested with restriction endonuclease.Plenty of studies have shown that MAR considered as initiaI point of DNA replication or transcription of regulatory gene.Thereby,construction of MAR expression vector can elevate the overall level of transgene expression,enhance stability of exogenous gene.as welI as increase frequency of stable transfectant cells.OBJECTIVE:To construction pLXSN-CAT recombinant retrovirus vector that containing chloramphenicol acetyltransferase(CAT)via cloning MAR sequence of human.and to explore the influence of MAR on the gene expression.METHODS:An open experiment was performed at the Department of Biochemistry and Molecular Biology.Xinxiang Medical College from September 2007 to December 2007 The PLXSN-CAT vector of CAT was constructed by the laboratory.TaqDNA polymerase,T_4 DNA ligase,DNA Marker,restriction enzyme BamH I,agarose gel DNA purification kit,as well as plasmid purification kit were purchased fromTakara Biotechn0Iogy(Dalian)Co.,Ltd.The sequence of csp-B MAR was amplified by polymerase chain reaction(PCR)method applied to human DNA.The fragment was inserted into retrovirus vector PLXSN-CAT plasmid.The recombinant plasmid was verified by double digestion and DNA sequencing.RESULTS AND CONCLUSION:The length of specific fragment applied by PCR was 931 bp,and the recombinant plasmid PLXSN-CAT-MAR presented two bands:5.9 kb and 931 bp using respective restriction enzymes BamH I The sequence of MAR was confirmed by blasting to Genbank(serial numobr:M6271 6).It suggested that MAR had been cloned into PLXSN-CATR vector correctly.The recombinant retrovirus vector PLXSN-MAR was successfully constructed.

Objective: To investigate the AIDS-related knowledge, attitudes and practice among university students in a pilot site of AIDS health education in Zhoushan City, so as to provide the reference for AIDS health education in universities. Methods: University students were recruited from Zhejiang Ocean University using a stratified cluster sampling method in 2018 and 2020, and the AIDS-related knowledge, attitudes and practice were collected using a questionnaire survey. Results: A total of 2 862 and 2 850 students were surveyed in 2018 and 2020, including 1 429 ( 49.93% ) and 1 414 ( 49.61% ) male students, respectively. The overall awareness of AIDS-related knowledge was 86.62% and 94.11% in 2018 and 2020, respectively. There were 544 ( 19.01% ) and 394 ( 13.82% ) students that were in favor of one-night stand, 308 ( 10.76% ) and 198 ( 6.95% ) students that were in favor of commercial sexual behaviors, and there were 59 ( 2.06% ) and 34 ( 1.19% ) students that had casual sexual behaviors within one year, and 20 ( 0.70% ) and 8 students ( 0.28% ) with commercial sexual behaviors within one year. There was a significant difference in the awareness of AIDS-related knowledge among students with different years at the university ( P<0.05 ). The lowest awareness of AIDS-related knowledge was seen in freshmen in 2018 ( 81.74% ), and the highest awareness was found in freshmen in 2020 (97.17% ). The proportions of being in favor of one-night stand ( 2018: 31.35% vs. 6.70%; 2020: 22.07% vs. 5.71%; P<0.05 ), being in favor of commercial sexual behaviors ( 2018: 19.91% vs. 2.23%; 2020: 12.09% vs. 1.88%; P<0.05), having casual sexual behaviors within one year (2018: 3.71% vs. 0.42%; 2020: 2.19% vs. 0.21%; P<0.05), and having commercial sexual behaviors within one year ( 2018: 1.33% vs. 0.07%; 2020: 0.50% vs. 0.07%; P<0.05 ) were significantly greater in male students than in female students. Conclusions The pilot AIDS health education is effective to increase the awareness of AIDS-related knowledge, possess the correct attitudes towards sexual behaviors and reduce high-risk sex behaviors among university students in Zhoushan City. Intensified AIDS health education is recommended among senior and male university students.

Liver cancer is one of the most common cancers,and its common surgical treatment methods include tran-scatheter arterial chemoembolization,radiofrequency ablation,and liver transplantation surgery.However,the treatment effect of these surgeries on patients with mid-to late-stage liver cancer is not ideal.In recent years,with the continuous development of tumor gene therapy and tumor immunology,tumor treatment methods have transitioned from traditional models to targeted onco-lytic virus therapy.With the advantages of fast replication,the oncolytic virus can kill tumor cells without damaging other normal cells and realize the targeted treatment of liver cancer through mechanisms such as activating the immune system and improving the tumor microenvironment.In addition,immunotherapy can reduce tumor recurrence and metastasis,thereby exerting therapeutic effects on liver cancer.This article reviews the research progress of oncolytic virus and immunotherapy for liver cancer,aiming to provide a reference for the clinical treatment of liver cancer.

OBJECTIVE: To explore the genetic etiology of a child with Pitt-Hopkins syndrome. METHODS: A child who had presented at the Medical Genetics Center of Gansu Provincial Maternal and Child Health Care Hospital on February 24, 2021 and his parents were selected as the study subjects. Clinical data of the child was collected. Genomic DNA was extracted from peripheral blood samples of the child and his parents and subjected to trio-whole exome sequencing (trio-WES). Candidate variant was verified by Sanger sequencing. Karyotype analysis was also carried out for the child, and her mother was subjected to ultra-deep sequencing and prenatal diagnosis upon her subsequent pregnancy. RESULTS: The clinical manifestations of the proband included facial dysmorphism, Simian crease, and mental retardation. Genetic testing revealed that he has carried a heterozygous c.1762C>T (p.Arg588Cys) variant of the TCF4 gene, for which both parents had a wild-type. The variant was unreported previously and was rated as likely pathogenic based on the guidelines of the American College of Medical Genetics and Genomics (ACMG). Ultra-deep sequencing indicated that the variant has a proportion of 2.63% in the mother, suggesting the presence of low percentage mosaicism. Prenatal diagnosis of amniotic fluid sample suggested that the fetus did not carry the same variant. CONCLUSION The heterozygous c.1762C>T variant of the TCF4 gene probably underlay the disease in this child and has derived from the low percentage mosaicism in his mother.
Child ; Female ; Humans ; Male ; Pregnancy ; Intellectual Disability/genetics* ; Mosaicism ; Mothers ; Mutation ; Parents ; Transcription Factor 4/genetics*