АНОТАЦІЯ
Мелешко Р.А. Одержання біологічно-активного пролактину людини з використанням бакуловірусної експресійної системи.– Рукопис.
Дисертація на здобуття наукового ступеня кандидата біологічних наук за спеціальністю 03.00.20 – біотехнологія. – Інститут молекулярної біології та генетики НАН України, Київ, 2005.
Дисертація присвячена вивченню експресії рекомбінантного пролактину людини з використанням бакуловірусної експресійної системи (БЕС) та розробці лабораторної методики одержання і очищення біологічно-активного рекомбінантного пролактину людини.
Вперше було синтезовано рекомбінантний PRL людини в бакуловірусній експресійній системі на основі ВЯП Malacosoma neustria (Mane). Сконструйовано рекомбінантні бакуловіруси, що забезпечують експресію внутрішньоклітинного та секретованого рекомбінантного пролактину людини в клітинах комах.
Відпрацьовано умови культивування клітин, інфікування їх вірусом, визначено оптимальний час після інфікування для збору клітин і середовища з метою досягнення максимальних рівнів експресії пролактину, розроблено методи очищення біологічно-активного рекомбінантного пролактину людини, що дозволяє отримати дві форми рекомбінантного гормону – внутрішньоклітинну і секретовану у кількостях 60 мг і 10 мг білка відповідно на один літр поживного середовища.
На базі проведених досліджень запропоновано лабораторну методику одержання біологічно-активного рекомбінантного очищенного пролактину людини.
Ключові слова: бакуловірусна експресійна система, пролактин, експресія.
АННОТАЦИЯ
Мелешко Р.А. Получение биологически-активного пролактина человека с использованием бакуловирусной эспресионной системы.– Рукопись.
Диссертация на соискание ученой степени кандидата биологических наук по специальности 03.00.20 – биотехнология. – Институт молекулярной биологии и генетики НАН Украины, Киев, 2005.
Диссертация посвящена изучению экспрессии рекомбинантного пролактина человека с использованием бакуловирусной экспрессионной системы (БЭС) и розработке лабораторной методике получения и очистки биологически-активного рекомбинантного пролактина человека.
Впервые был синтезирован рекомбинантный PRL человека в бакуловирусной єкспрессионной системе на основе ВЯП Malacosoma neustria (Mene). Сконструировано рекомбинантные бакуловирусы, которые обеспечивают экспрессию внутриклеточного и секретируемого пролактина человека в клетках насекомых.
Отработаны условия культивирования клеток, инфицирования их вирусом, определено оптимальное время после инфицирования для сбора клеток и среды с целью достижения максимальных уровней экспрессии пролактина, разработаны методы очистки биолоически-активного рекомбинантного пролактина человека, что позволяет получить две формы рекомбинантного гормона – внутриклеточную и секретируемую в количествах 60 мг и 10 мг белка соответственно на один литр питательной среды.
На базе проведенных исследований предложена лабораторная методика получения биологически-активного рекомбинантного очищенного пролактина человека.
Ключевые слова: бакуловирусная экспрессионная система, пролактин, экспрессия.
SUMMARY
Meleshko R.A. Production of human biologically active prolactin by using baculovirus expresson system. – Manuscript.
Thesis for a Philosophy Doctor (Ph.D.) degree in Biology by speciality 03.00.20 – biotechnology. – Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine, Kyiv, 2005.
The thesis is devoted to the elaboration of expression of human prolactin by using baculovirus expresson system with following purification of biologically active recombinant hormone.
Prolactin is a polypeptide hormone secreted by the pituitary gland. PRL regulates multiple physiological functions. Prolactin present in mammals in several forms is heterogenous and apparently is a subject of posttranslational modifications including glycosilation, phosphorylation, proteolytic cleavage, and amidation. This was the reason for choosinge the baculovirus expression system since this system produces foreign protein in large quantity and, in most cases, the recombinant proteins are processed and modified in a manner similar to that of their authentic counterparts.
The effective expression of human recombinant prolactin has been reached by using of baculovirus expression system on the basis of: Malacosoma neustria nuclear polyhedrosis virus (NPV) – Antheraea pernyi insect monolayer cell culture. This system was elaborated in IMBiG NASU as a prospective alternative of other commonly used baculovirus expression system. But the disadvantage of Malacosoma neustria as a vector is impossibility to use different cellular lines for its production due to restricted host range for this virus.
During past 10-15 years the most commonly baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) system was used. For this particular system a wide spectrun of convenient transfer vectors was developed, making cleaning of proteins synthesized in this system relatively simple and easy. We used for expression of human recombinant prolactin recombinant viruses АcMNPV and Hf, Sf9, Sf21 insect monolayer cell cultures. Recombinant АcMNPV – Hf cells system demonstrated the highest level of prolactin expression (245 mg/L), due to this fact it was chosen for further work on the way to production of different forms of recombinant prolactin.
Several recombinant Autographa californica nuclear polyhedrosis virus vectors were designed:
1. AcHisPRL vector, which contains cDNA human prolactin with the nucleotide sequence on the 5' terminus that encodes 6 histidine residues for the intracellular chimeric prolactin expression.
2. AcMelPRL vector, which contains cDNA of human prolactin with the nucleotide sequence on the 5' terminus that encodes the signal peptide of bee melitin for the secreted prolactin expression.
3. AcMelPRLHis vector, which contains cDNA of human prolactin with the nucleotide sequence on the 3'-terminus that encodes 6 histidine residues and with the nucleotide sequence on the 5'-terminus that encodes the signal peptide of bee melitin for the secreted chimeric prolactin expression.
It was demonstrated decreased biological activity of His-containing secreted prolactin (MelPRLHis) and instability of its expression levels in insect cells. Due to these facts it was concluded that production of this form of hormone wasn’t prospective.
The HF cells provided the high stable levels of expression of both intracellular (HisPRL) and secreted (MelPRL) prolactin. The positive influence of the secreted prolactin on the growth of host HF cells was mentioned. The cells of this line were adapted to the suspension cultivation. It was observed an approximately twofold increase of the recombinant hormone production in suspension culture1 (both for the intracellular and secretory prolactin forms) comparing with its production in monolayer culture.
Additional attempts were made to provide conditions for the most efficient prolactin expression: influence of MOI of infection and postinfection time for cell harvesting on the levels of prolactin expression was investigated. Combination of conditions, which provide the maximum yield of the recombinant product were established: HF cell suspension cultivation, infecting them with the MOI 1, harvesting prolactin containing cells and medium after 48 hours post infection.
Under conditions used recombinant prolactin expression levels were estimated as 500 mg/L and 20-30 mg/L for intracellular and secreted prolactin forms respectively.
The recombinant protein purification methods were suggested. The use of AcHisPRL for cell infection enable us to introduce simple quick and easy purification method based on Talon metal affinity resin.
Two purification methods for intracellular protein were used: purification using metall-affine chromatography under denaturing conditions with the subsequent renaturing of recombinant protein; prolactin denaturing with the subsequent renaturing and purification of renatured protein using metall-affine chromatography under the native conditions.
The heparin-affinity chromatography method was used for purification of secreted prolactin.
95% of purity of recombinant protein was reached, the quantity of the purified product was estimated as 60 mg of the intracellular protein per 1 L of medium and 10 mg of the secreted protein per 1 L of medium. It was shown that approximately 50% of secreted prolactin was glycosylated.
The estimation of the activity of expressed protein was performed. The biological activity of secreted and intracellular prolactin forms testing by proliferation assay with Nb2 lymphoma cells and appeared to be approximately equal and correspondented the biological activity of commercial nonrecombinant original human prolactin, used as control in Nb2 cells routinely appield for hormone estimation medical tests.
Based on the performed research the method of expression and purification of biologically active human prolactin was suggested. The baculovirus expression system described here can be used for large-scale producrion of bioactive recombinant human prolactin for scientific purpose and for medical diagnosticum test systems.
Key words: baculovirus expresson system, recombinant prolactin.
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