Construction of Marker-Free GFP Transgenic Tobacco by Cre/lox Site-Specific Recombination System

Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation. The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation. After removal of the Cre-NPT Ⅱ locus by genetic segregation through self-cross, plants that incorporated only the GFP transgene were obtained. Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently introduced into 5 plants of them, respectively, by retransformation. The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT. The results showed that few discs were able to regenerate normally, and the excision at 76-100% efficiency depended on individual retransformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration of any submarginal nucleotides of the recombination sites. Bar gene excised plants were selfpollinated to allow segregation of the GFP gene from the Cre-NPT Ⅱ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPT Ⅱ locus in the Kan senstive plants were confirmed by PCR analysis subsequently. Hence, constructing marker-free transgenic tobacco plants by Cre/lox sitespecific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.

Site-specific recombination in Escherichia coli mediated by actinomyces phage R4 integrase

The purpose of this study was to demonstrate that actinomyces phage R4 integrase Sre protein efficiently mediate site-specific recombination in Escherichia coll. An intramolecular recombination assay system in E. coli was constructed. The plasmid pBZP contains attB and attP sites in direct orientation flanking a lacZ gene. When pBZP was introduced into E. coli cells, in which the plasmid pSREA containing sre gene was resident, Sre protein catalyzed integration of attP into attB site, resulting in excision of the lacZ gene. This integration changed bacteria colonies from blue to white on agar plates containing X-Gal, which showed that the lacZ was removed. The integrant DNAs were identified by enzyme digestion, PCR and DNA sequencing. The minimal sizes of attB and attP were 50 bp and 47 bp for 100% recombination efficiency. The phage recombinase Sre efficiently integrated attP into attB site to create attR and attL in E. coli host environment without Streptomyces specific cofactors. This intrmolecular assay system is a simple and efficient system for Sre-mediated recombination in E. coll.

Skin care efficacy study of recombinant humanized collagen based on in vitro level

Studying the skin care efficacy of recombinant humanized collagen based on in vitro level.The stability of the recombinant humanized collagen was first analyzed by treating at different temperatures,then its skincare efficacy based on in vitro level was evaluated by detecting the inhibition rate of elastase,the inhibition rate of collagenase,the protein content of type I collagen in human fibroblasts,the inhibition of reactive oxygen species(ROS)with human keratinocytes,and the effects of the recombinant humanized collagen on the expression of hyaluronic acid(HA),filaggrin(FLG)and transglutaminase 1(TGM1)in keratinocytes.The results showed that recombinant humanized collagen was able to maintain stability at temperatures below 70℃.With regard to its skincare efficacy,recombinant humanized collagen could inhibit elastase and collagenase activities and promote the increase of type I collagen content in human fibroblasts.It also showed good inhibition of ROS in keratinocytes in vitro and could increase the expression of HA,FLG,and TGM1 in keratinocytes.In short,the recombinant humanized collagen exhibited a favourable skin care effect in vitro level.This study proved that it has potential firming,anti-wrinkle,moisturizing,and repairing efficacy,and is a valuable cosmetic raw material.

Recombinant amelogenin regulates the bioactivity of mouse cementoblasts in vitro

Amelogenin（AMG） is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to investigate the effects of recombinant human AMG（rhAMG） on mineralized tissue-associated genes in cementoblasts. Immortalized mouse cementoblasts（OCCM-30）were treated with different concentrations（0.1, 1, 10, 100, 1 000, 10 000, 100 000 ng · mL^-1） of recombinant human AMG（rhAMG）and analyzed for proliferation, mineralization and mRNA expression of bone sialoprotein（BSP）, osteocalcin（OCN）, collagen type I（COL I）, osteopontin（OPN）, runt-related transcription factor 2（Runx2）, cementum attachment protein（CAP）, and alkaline phosphatase（ALP） genes using quantitative RT-PCR. The dose response of rhAMG was evaluated using a real-time cell analyzer.Total RNA was isolated on day 3, and cell mineralization was assessed using von Kossa staining on day 8. COL I, OPN and lysosomalassociated membrane protein-1（LAMP-1）, which is a cell surface binding site for amelogenin, were evaluated using immunocytochemistry. F-actin bundles were imaged using confocal microscopy. rhAMG at a concentration of 100 000 ng · mL^-1 increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG（100 000 ng · mL^-1） upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100 000 ng · mL^-1 remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10 000 and 100 000 ng · mL^-1 rhAMG. The present data suggest that rhAMG is a potent regulator of gene expression in cementoblasts and support the potential application of rhAMG in therapies aimed at fast regeneration of damaged periodontal tissue.

Recombinant human bone morphogenetic protein-7 expressed from CHO cells possessing the activity of bone-induced in vitro

Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (CHO) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to CHO cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this CHO expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.

Overview of Recombinant Skin Models and Progress in Their Application in Vitro Assessment of Toxicity and Efficacy

A recombinant skin model is a model in which skin cells are grown in vitro on a bioactive scaffold and provided with adequate nutrition to promote cell proliferation and differentiation to produce a mock skin structure and biological features. The development of recombinant skin models allows for effective and scientifically sound in vitro evaluation tests. This review briefly summarizes the overview of recombinant skin models and the progress of their application in in vitro evaluation which focuses on three aspects: skin irritation, skin corrosivity, and anti-skin aging. Moreover, an outlook on the future development of recombinant skin models is also provided in this review.

Establishment and application of both FLP and Cre site-specific recombination systems at the same position in the genome

Both FRT-FRT and LoxP-LoxP sites that are the target sepuences of site-specific recombinases have been constructed in a vector, called C4LFY, using the recombinant DNA technigue. C4LFY also contains P elements, 2 exons and 1 intron of Drosophila yellow gene, yellow promoter and enhancers, and flanking DNA. Since C4LFY made use of two pairs of FRT and LoxP sites, this vector included two site-specific recombination systems. C4LFY was then integrated into Drosophila genome by P-element-mediated germ line transformation. in the presence of the FLP or Cre recombinase, either FLP/FRT or Cre/LoxP recombination reaction was successfully created at the same position in the genome. Using this system, the molecular basis of yellow gene expression and regulation during development have been investigated. Results indicate that the tissue-specific expression of yellow gene is directly regulated by transcriptional enhancers. in addition, the 5’ and 3’ genomic sequences flanking the yellow gene have been

Co-transformation to tobacco of Cre/lox site-specific recombination system and its precise recombination

For the temporally and spatially regulated expression of the barnase gene in plant, two kinds of plasmids with ere gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure. The transgenic plants were conformed by PCR analysis. The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome. Cloning and seguencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre//ox site-specif ic recombination .

The development of a recombinant hybrid protein of Plasmodium falciparum and analysis of its antigenicity and protectivity

A hybrid gene encoding several putative protective epitopes from erythrocytic stage antigens (MSA1,MSA2 and RESA) of P. falciparum as well as exgenous T cell enhancer epitopes from interleukin-1 and tetanus toxin was synthesized chemically. This gene (named HGFC) was cloned and connected with another hybrid gene (HPFA) synthesized previously to make a bigger hybrid gene (HGFCAC). HGFC and HGFCAC were cloned in an expression vector pWR450-1 and transformed into E. Coli JM109. The engineered bacteria could express fusion proteins with molecular weights of 65 and 77 kDa after inducing with isopropylthio-β-D-galactoside (IPTG). The expression rate was about 35% of total bacterial proteins. The expressed products showed sepcific immunological reaction with rabbit antibodies against P. falciparum peptide Glu-Glu-Asn-Val-Glu-His-Asp-Ala (EENVEHDA)by Western blotting. The fusion proteins were pruified by precipitation with amonium sulfate. gel filtration and ion-exchange chromatography and the purity was 82%. The purified protein reacted specifically with mouse immune serum against falciparum blood stage antigens by dot enzyme-linked immunosorbent assay (dot-ELISA).The fusion protein was emulsified with Freund's complete adjuvant (FCA) and used to immunize rabbits. The immune serum can recognize P. falciparum erythrocytic stage antigens of Fcc-1/HN strain and Yunnan strain and had weak cross reaction with P. vivax,but had no reaction with P. cynomlogi and P. berghei antigens. The protective effect of the antibody was tested by in vitro inhibition test to cultured falciparum parasites. Preliminary results indicated that the immune sera could effectively reduce the invasion rates of the parasites to red blood cells and inhibit the growth of the in vitro cultured falciparum parasites. The inhibitory capacity of the immune sera to parasite invasion is enhanced as the amount of the sera increases and the incubation time of the sera with the parasites is prolonged.It was shown that after 72 h incubation at 20% concentration with the parasites, the serum can suppress the multiplication of P.falciparum growth in vitro to a level of 80%.The immune sera caused dispersion of the parasite cytoplasm,atrophy of parasites,agglutination of free merozoites and degeneration of schizonts.

The Effect of Recombinant LH Addition to Recombinant FSH on Assisted Reproductive Technologies Outcomes in Overweight and Obese Patients without Polycystic Ovary Syndrome

Background: Obesity and overweight negatively affect fertility. Protocols need?to be developed in ART treatments for obese and overweight women. Objectives: To investigate the effect of recombinant luteinizing hormone (rLH) addition to recombinant follicle stimulating hormone (rFSH) on treatment outcomes?in assisted reproductive technologies (ART) cycles in obese and overweight women without polycystic ovary syndrome. Methods: This retrospective cohort study was carried out in Kocaeli University Faculty of Medicine, Assisted Reproductive Techniques Clinic between January 2016 and March 2019. To analyze the impact of rLH addition to rFSH in GnRH antagonist cycles in overweight and obese, the patients were divided into four groups according to body mass index (BMI) and gonadotropin type;Group 1: patients with BMI ≥ 25 stimulated with rFSH alone (n: 37), Group 2: patients with BMI ≥ 25 stimulated with rFSH + rLH (n: 37), Group 3: patients with BMI between 18.5 - 24.99 stimulated with rFSH alone (n: 33), Group 4: patients with BMI between 18.5 and 24.99 stimulated with rFSH + rLH (n: 30). Patients with polycystic ovary syndrome were excluded. Results: Basal LH levels were found to be significantly lower in obese and overweight patients compared to normoweight patients (p = 0.01). Grade 1 embryo ratio in obese and overweight patients was higher in rLH added obese group than in group LH not included (64.9%, p = 0.005). Ongoing pregnancy rates (OPR) in obese and overweight patients were significantly higher in rLH added group compared to rFSH only group (43.2% vs 18.9% respectively, p = 0.044). However, OPR did not differ significantly in rLH added and rFSH only groups in normoweight patients (p = 0.588). Conclusion: This study has shown that obese and overweight non-PCOS patients have lower endogenous LH levels. It has also shown that rLH supplementation in GnRH antagonist cycles in obese and overweight women improves embryo quality and ongoing pregnancy rates. However rLH addition to rFSH doesn’t seem to have a value in normoweight patients.

PPOS方案促排卵过程中添加重组人生长激素注射液对卵巢低反应患者IVF结局的影响

目的探讨在卵泡期高孕激素状态下促排卵(PPOS)方案中联合使用重组人生长激素注射液对卵巢低反应患者体外受精-胚胎移植结局的影响。方法选取2020-2022年在江西省妇幼保健院辅助生殖中心进行助孕治疗的患者2137例,按照是否在PPOS过程中使用重组人生长激素注射液分为试验组(n=730)和对照组(n=1407)。对2组的临床特征及治疗结局进行回顾性PSM分析。结果试验组获卵数(3.87±2.93 vs3.21±2.44,P<0.001)、可移植胚胎数(1.47±1.18vs1.31±1.12,P=0.008)、HCG阳性率(64.0%vs52.7%,P=0.004)均显著高于对照组。结论PPOS方案中联合使用重组人生长激素注射液可增加卵巢低反应患者获卵数,显著改善卵母细胞和胚胎的数量及质量,改善临床结局。

重组卵泡刺激素预充注射笔在控制性超促排卵中的临床应用

目的探讨重组卵泡刺激素预充注射笔在中国人控制性超促排卵中的效果与可行性。方法对南方医科大学南方医院生殖医学中心2007.01～2008.04间就诊的184例需行体外受精-胚胎移植(IVF-ET)不孕症妇女,采用黄体期长方案,随机分别给予重组卵泡刺激素预充注射笔(A组)和重组卵泡刺激素冻干粉制剂(B组)进行卵巢刺激,分析比较两组间用药情况、促排卵与IVF-ET结局。结果A组rFSH总用量、前往医疗单位次数、购药和注射费用明显少于B组(P<0.05);在用药时间上没有显著性差异(P>0.05);A组剩余药量明显高于B组(P>0.05)。A组用药局部红肿的比例显著少于B组(P<0.05),但用药局部瘀斑的比例明显多于B组(P<0.05)。A组药物易用性和方便性评分均显著高于B组(P<0.05);两组在疼痛感受上没有显著性差异(P>0.05)。A组进行了更频繁的药物个体化调整(P<0.05),而其hCG日血清E2浓度明显低于B组(P<0.05)。但两组hCG日内膜厚度、总获卵数、第二次减数分裂中期卵子数量、每周期移植胚胎数以及临床妊娠率没有显著性差异(P>0.05)。A组成熟卵子比例、优质胚胎率和胚胎着床率明显高于B组(P<0.05),而发生中重度OHSS的例数明显少于后者(P<0.05)。结论重组卵泡刺激素预充注射笔具有高效、安全、易用、价廉和患者耐受性好等优点,有利于个体化COS治疗方案的实施,在临床推广中应注意合理选择和搭配制剂种类并加强对患者的健康教育。

重组人纽兰格林生物学活性检测方法及质控标准

目的 研究重组人纽兰格林生物学活性检测方法 ,并制定其质量控制标准。方法 采用激酶受体活化的酶联免疫吸附试验 (KIRA ELISA) ,通过优化细胞浓度、样品稀释梯度、药物刺激时间及细胞裂解方法等建立定量测定重组人纽兰格林体外生物学活性的方法 ,同时建立了一套完整可行的质量控制标准。结果 重组人纽兰格林刺激MCF 7细胞表面ErbB2 ErbB3分子磷酸化的反应存在量 效关系 ,相关系数大于 0 98,原液的平均比活性为 85 10U mg± 115 0U mg。并对原液设立肽图、纯度等检测项目 ,对成品设立热原质试验、鉴别试验等检测项目。结论 KIRA ELISA可用于定量检测重组人纽兰格林的体外生物学活性 ,结果准确可靠 ,重复性好 ;建立的方法和标准可以有效控制重组人纽兰格林制品的质量。

体外培养和冷冻保存对微囊化细胞生长和内皮抑素表达的影响

微囊化基因工程细胞移植治疗肿瘤是一种新兴的肿瘤治疗方法,如果将此技术应用到临床研究,就需要制备大量的细胞活性良好、重组蛋白表达量高的生物微胶囊。体外培养和冷冻保存是生物微胶囊制备过程中两个重要的环节,因此需要考察体外培养和冷冻保存对微囊化重组基因细胞生长和蛋白表达的影响。以重组CHO细胞为模型,考察了体外培养时间和冷冻保存对微囊化细胞在动物体内生长和内皮抑素表达的影响及体外培养时间对微囊化细胞冷冻保存的影响。结果表明:体外培养时间对微囊化细胞在动物体内生长、内皮抑素表达和微囊稳定性具有较大的影响,体外不培养和培养4d的微囊化细胞在小鼠腹腔内生长良好、内皮抑素表达量高,并且微囊稳定性好,而体外培养8d的微囊化细胞在移植后的第26天破裂。体外培养时间对微囊化细胞冷冻保存也具有较大的影响,体外培养4d和8d的微囊化细胞在液氮中冷冻保存40d,复苏后细胞生长良好、内皮抑素表达量高,而冻存前未经过体外培养的微囊化细胞,复苏后细胞几乎全部死亡。综上所述,生物微胶囊在体外比较适宜的培养时间为4d。并且冷冻保存对微囊化细胞在动物体内生长、内皮抑素表达和微囊稳定性没有显著的影响。

干扰素诱导Mx-Cre转基因小鼠表达的重组酶活性的体外检测

利用ＰＣＲ、Ｗｅｓｔｅｒｎ ｂｌｏｔ、免疫组化、免疫金标电镜、Ｓｏｕｔｈｅｒｎ ｂｌｏｔ从ＤＮＡ水平、蛋白水平分析干扰素诱导后Ｍｘ－Ｃｒｅ转基因小鼠肝组织中Ｃｒｅ重组酶的表达及其表达产物的活性。在对Ｍｘ－ｃｒｅ转基因小鼠基因组中整合有ｃｒｅ基因进行确定后，通过干扰素诱导Ｍｘ－ｃｒｅ转基因小鼠表达Ｃｒｅ重组酶，结果表明转基因小鼠肝细胞核和细胞质中均有Ｃｒｅ重组酶的表达，并在超微水平进一步证实。将含表达的Ｃｒｅ重组酶的肝细胞核抽提液加入到带有ｌｏｘＰ位点的ＤＮＡ中进行重组，分析证明Ｍｘ－Ｃｒｅ转基因小鼠表达的Ｃｒｅ重组酶具有重组活性，从而建立了体外检测Ｍｘ－Ｃｒｅ转基因小鼠表达的Ｃｒｅ重组酶活性的方法。

我国D2-43病毒株PrM-E基因的重组SFV的制备及生物学鉴定

为构建含PrM E基因的重组SFV病毒 ,将含我国登革 2型病毒PrM E基因的SFV表达载体和辅助载体DNA线性化后 ,进行体外转录 .然后将获得的 2种RNA转录物共转染BHK2 1细胞 ,以RT PCR法证明转染后的细胞培养上清中含有重组SFV .进一步将该重组病毒经蛋白酶激活后可使BHK2 1细胞产生细胞病变 ,并且以间接免疫荧光法在感染细胞内可检测到登革 2型病毒所特有的蛋白 .含我国登革 2型病毒PrM E基因的重组SFV的获得 ,为进一步观察该重组病毒的免疫原性奠定了基础 。

猪第一极体核物质参与卵母细胞受精及其胚胎发育的研究

旨在探索猪卵母细胞第一极体核物质能否参与体外受精及其进一步的胚胎正常发育。第一极体(FPB)获自猪卵巢卵泡中卵丘卵母细胞复合体(COCs)体外成熟培养的MⅡ期卵母细胞。存活极体从成熟卵母细胞内分离后采用显微操作术注入去核卵母细胞胞质中。第一极体重组卵母细胞分别采用常规体外受精和单精子胞浆注射法进行受精,来自极体核重组的受精卵进行体外培养,观察和分析其进一步体外卵裂发育的能力。结果显示:极体显微操作重组的成功率约为80%,20%左右的重组卵母细胞于重组后的受精和发育培养过程中退化、崩解;常规体外受精和单精子注射后2-细胞卵裂率分别达到12.9%和29.8%。结论:猪第一极体核物质具有参与卵母细胞受精和进一步胚胎发育的能力。据此推测利用第一极体将有可能繁殖正常仔猪后代。

重组人红细胞生成素体内外活性检测方法的比较

目的对重组人红细胞生成素 (rhEPO)体内外活性测定结果进行比较 ,说明体内外活性测定对rhEPO质量控制的意义。方法采用59Fe摄入法、网织红细胞法和ELISA法测定rhEPO制品的体内外生物学活性。结果59Fe摄入法和网织红细胞法测定结果一致 ,与rhEPO唾液酸含量具有相关性。当rhEPO唾液酸含量较低时 ,ELISA法与体内活性测定结果有较大差异。结论rhEPO体内外活性测定方法可以相互补充 ,均是保证rhEPO质量不可缺少的质控手段。

重组人生长激素缓释微球.Ⅰ.制备与体外释放研究

采用复乳-溶媒蒸发法制备重组人生长激素(rhGH)可生物降解缓释微球,考察蛋白稳定剂泊洛沙姆407(F127)对rhGH体外释放的影响。利用均匀设计优化微球的处方和制备工艺,制得的rhGH-PLGA微球球形圆整,表面光滑致密,粒径均匀且具有较高的包封率和载药量。微球的体外释药行为呈先快后慢的两相释放模式,d30时含有F127的微球累积释药量约79%,较不含稳定剂的微球高22%。表明F127可通过抑制有机溶剂诱导的蛋白聚集而对rhGH起到稳定作用。

枯草杆菌蛋白酶基因工程的研究进展

本文介绍了枯草杆菌蛋白酶 (Subtilisin)的研究现状 ,即利用定位诱变和体外重组等技术改变酶的性质 ,包括催化活性、底物特异性、稳定性、低温适应性以及酶在有机相中的性能等。对枯草杆菌蛋白酶的成功改造不仅有可观的商业价值 。