结核分枝杆菌重组质粒pGEX-ESAT-6构建及其在大肠埃希菌BL21(DE3)中表达效率的研究

目的构建结核分枝杆菌（MTB）重组质粒pGEX-ESAT-6，分析ESAT-6抗原在大肠埃希菌BL21（DE3）中的表达效率。方法以结核分枝杆菌H37Rv标准株基因组DNA为模板，通过PCR扩增得到ESAT-6抗原编码基因；将该基因定向克隆于含有谷胱甘肽-S-转移酶（GST）基因的高效原核表达载体pGEX-1λT，经酶切鉴定后以IPTG诱导表达ESAT-6／GST融合蛋白；SDS—PAGE及Western blot对表达产物进行鉴定。结果PCR扩增出288bp的ESAT-6基因；双酶切证实ESAT6基因成功插入pGEX-1λT中，构建了pGEX—ESAT-6穿梭表达载体。SDS—PAGE分析重组质粒pGEX-ESAT-6表达产物的分子质量单位为35ku，表达效率为20％，Western blot检测该蛋白能被活动性结核病人血清特异识别。结论结核分枝杆菌重组质粒pGEX-ESAT-6在大肠埃希菌中获得了高效融合表达，表达的ESAT-6重组蛋白具有抗原特异性。     

日本血吸虫重组质粒pQE30-Sj26GST-Sj32的构建及其在大肠埃希菌BL21(DE3)中的表达

目的构建日本血吸虫重组质粒pQE30-Sj26GST-Sj32,研究该质粒在大肠埃希菌BL21(DE3)中的表达。方法超声粉碎日本血吸虫成虫,提取总RNA,通过RT-PCR扩增获Sj26GST和Sj32抗原编码基因;采用基因拼接法(gene SOEing)剪接Sj26GST和Sj32,得到Sj26GST-Sj32融合基因;将融合基因克隆至原核表达载体pQE30,构建重组质粒pQE30-Sj26GST-Sj32并转化大肠埃希菌BL21,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果基因拼接法扩增出约1 991 bp的Sj26GST-Sj32融合基因;双酶切和PCR鉴定证实Sj26GST-Sj32融合基因成功插入pQE30中,SDS-PAGE显示表达产物为分子质量单位约为61 ku的重组蛋白,与预期结果一致,表达的蛋白约占菌体总蛋白的18%;Western blot鉴定重组蛋白能被日本血吸虫感染的兔血清识别。结论成功构建了日本血吸虫重组质粒pQE30-Sj26GST-Sj32,该质粒在大肠埃希菌BL21中获得了高效融合表达,表达的融合蛋白具有抗原特异性。     

弓形虫关键粘附蛋白在大肠埃希菌BL21（DE3）中的表达

目的通过cDNA文库筛选出弓形虫关键粘附蛋白基因一棒状体ROP2基因,在大肠埃希菌BL21（DE3）中表达,以获得重组蛋白。方法根据弓形虫棒状体ROP2基因开放阅读框设计引物,以弓形虫cDNA为模板进行PCR,纯化扩增产物经双酶切,进行TA克隆,再克隆到pET-30a中。重组质粒pET-30a—top2经PCR鉴定后,转化到大肠埃希菌BL21（DE3）中,经IPTG诱导表达,用SDS—PAGE电泳鉴定表达产物。结果获得的1223bp的基因片段经PCR和酶切鉴定后与预期大小一致;SDS—PAGE电泳分析显示诱导菌在52ku处出现一条明显的蛋白带,与预期分子量相符。结论成功构建PET30a—ROP2,并在大肠埃希菌BL21（DE3）中高效表达,为进一步研究弓形虫粘附关键蛋白基因与肠上皮细胞的关系奠定基础。     

细粒棘球绦虫亲环蛋白基因的克隆 重组表达和生物信息学分析

目的 克隆表达细粒棘球绦虫亲环蛋白 （EgCyP） 基因, 并对其进行生物信息学分析。方法 从细粒棘球绦虫cD? NA中扩增目的基因, 克隆入表达载体pET28a, 转化大肠埃希菌 （E.coli） BL21 （DE3）, 经异丙基?β?D硫代半乳糖苷诱导表达后, 进行SDS?PAGE和免疫印迹试验鉴定, 并对其进行生物信息学分析。 结果 重组质粒pET28a?EgCyP构建成功。SDS? PAGE和免疫印迹试验结果显示, 重组蛋白在E.coli BL21 （DE3） 中获得高效表达, 重组蛋白EgCyP分子量约为22 kDa, 可被细粒棘球绦虫感染犬血清识别。生物信息学分析显示该蛋白具有7个潜在的抗原表位。 结论 成功克隆出细粒棘球绦虫EgCyP基因并在E.coli BL21 （DE3） 中表达, 为进一步研究其免疫原性奠定了基础。     

肠出血性大肠埃希菌O157：H7外膜蛋白A的表达、鉴定和纯化

目的构建肠出血性大肠埃希菌O157：H7外膜蛋白A基因重组质粒，研究其在大肠埃希菌中的表达情况。方法用RT-PCR法从肠出血性大肠埃希菌O157：H7菌株克隆OmpA基因，构建重组质粒pET-30a（＋）-OmpA，经双酶切及基因测序鉴定后在E．coliBL21（DE3）原核表达系统中诱导OmpA的表达，通过蛋白质电泳技术检测蛋白的表达，用Western blot法测定和分析免疫反应性。结果获得的目的基因为1041bp，与预期值相符，测序结果与GenBank公布序列的同源性达100％，重组质粒pET-30a（＋）-OmpA在原核系统下可表达OmpA，Westernblot分析His-Tag单抗能与OmpA（His-Tag）发生免疫反应。结论OmpA重组质粒构建成功，表达产物具有抗原性。     

日本血吸虫重组质粒pET28α-Sj32的构建及其在大肠埃希菌BL21(DE3)中的表达

目的构建日本血吸虫重组质粒pET28α-Sj32,研究该质粒在大肠埃希菌BL21(DE3)中的表达。方法超声粉碎日本血吸虫成虫,提取总RNA,通过RT-PCR扩增Sj32抗原编码基因;将该基因克隆至原核表达载体pET28α,构建重组质粒pET28α-Sj32并转化大肠埃希菌BL21(DE3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果RT-PCR扩增出1270bp的Sj32基因;双酶切和PCR鉴定证实Sj32基因成功插入pET28α中,SDS-PAGE分析显示表达产物为分子质量单位约为42ku的重组蛋白,与预期结果一致,表达的蛋白约占菌体总蛋白的24%;Western blot鉴定重组蛋白能被日本血吸虫感染的兔血清识别。结论成功构建了日本血吸虫重组质粒pET28α-Sj32,该质粒在大肠埃希菌BL21中获得了高效融合表达,表达的融合蛋白具有抗原特异性。     

肠出血性大肠埃希菌O157∶H7外膜蛋白A的表达、鉴定和纯化

目的 构建肠出血性大肠埃希菌O157∶H7外膜蛋白A基因重组质粒,研究其在大肠埃希菌中的表达情况. 方法 用RT-PCR法从肠出血性大肠埃希菌O157∶H7菌株克隆OmpA基因,构建重组质粒pET-30a (+)-OmpA,经双酶切及基因测序鉴定后在E.coli BL21(DE3)原核表达系统中诱导OmpA的表达,通过蛋白质电泳技术检测蛋白的表达,用Western blot法测定和分析免疫反应性. 结果 获得的目的 基因为1 041 bp,与预期值相符,测序结果与GenBank公布序列的同源性达100%,重组质粒pET-30a (+)-OmpA在原核系统下可表达OmpA, Western blot分析His-Tag单抗能与OmpA(His-Tag)发生免疫反应. 结论 OmpA重组质粒构建成功,表达产物具有抗原性.     

铜绿假单胞菌重组质粒pGEX-OprF的构建及其表达

目的构建并鉴定铜绿假单胞菌重组质粒pGEX-OprF,研究该质粒在大肠埃希菌BL21(DE3)中的表达。方法以铜绿假单胞菌PAOl标准株总RNA为模板,自行设计引物,采用RT-PCR方法扩增OprF抗原编码基因,经酶切、连接后定向克隆入大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT,构建重组质粒pGEX-OprF,转化E.coliBL21(DE3)感受态细胞,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果 RT-PCR扩增出1 016 bp的OprF编码基因;重组质粒经双酶切和测序鉴定证实OprF基因成功插入pGEX-1λT中;SDS-PAGE分析表达产物分子质量单位约为61 ku,与预期结果一致,表达的蛋白质占菌体总蛋白的16%;Western blot鉴定重组蛋白能被Pa外膜粗抗原免疫小鼠血清识别。结论成功构建了铜绿假单胞菌重组质粒pGEX-OprF,该质粒在E.coliBL21(DE3)中高效融合表达,表达的融合蛋白具有抗原特异性。     

弓形虫RH株膜表面抗原2全长基因的高效表达与抗原性分析

目的　构建原核重组表达质粒pET23aSAG2,并在大肠埃希菌中实现高效表达,以及检测表达产物的抗原性。　方法　PCR扩增SAG2编码基因目的片段,琼脂糖凝胶电泳回收纯化,克隆至pMD18T载体,转化大肠埃希菌DH5α。测序后亚克隆至表达质粒载体pET23a,构建重组表达质粒pET2 3aSAG2,转化大肠埃希菌DH5α。筛选阳性克隆,经限制性酶切分析鉴定后,转化大肠埃希菌BL21(DE3),以异丙基 βD硫代半乳糖苷诱导表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS PAGE)与免疫印迹分析表达产物。　结果　PCR扩增出约500bp的SAG2编码基因目的片段,与预期片段大小相符,经测序鉴定无基因突变;所构建的pET23aSAG2重组表达质粒阳性克隆经PCR与双酶切鉴定,与预期结果一致;含有pET23aSAG2重组质粒的大肠埃希菌BL21(DE3)诱导后得到了高效表达,SDS PAGE显示表达产物约Mr19000;免疫印迹结果表明表达产物具有良好的抗原性。　结论　成功构建了pET23aSAG2表达质粒,实现了全长成熟SAG2蛋白在大肠埃希菌中的高效表达;表达产物具有良好的抗原性。     

新型冠状病毒Nsp16蛋白的原核表达及多克隆抗体的制备北大核心CSCD

目的原核表达新型冠状病毒Nsp16重组蛋白并制备兔抗Nsp16多克隆抗体。方法通过琼脂糖凝胶电泳对含有Nsp16基因的重组质粒进行双酶切鉴定,并将鉴定后的重组质粒转化大肠埃希菌BL21(DE3)感受态细胞;通过IPTG诱导重组蛋白的表达,利用镍柱亲和层析法纯化该重组蛋白并进行Western blot鉴定;将鉴定后的目的蛋白与弗氏佐剂以1:1的体积比混匀,乳化后免疫新西兰大白兔,利用ELISA和Western blot分别进行多克隆抗体效价的测定及其特异性鉴定。结果Nsp16基因重组质粒经双酶切后得到912bp的目的片段,重组质粒转化大肠埃希菌表达出分子质量单位为33.3ku的重组蛋白,且主要存在于上清中。用纯化的重组蛋白免疫大白兔,获得兔抗Nsp16特异性多克隆抗体,且其效价达1:409600以上。结论重组Nsp16蛋白可以在大肠埃希菌中正确表达,免疫大白兔后获得高效价抗Nsp16多克隆抗体,即具有免疫反应性。为SARS-CoV-2Nsp16蛋白在新型冠状病毒肺炎中的进一步研究奠定了基础。     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.