Separation, Purification And Identification Of A Protein Related With The Galactose-induced Cataract

Cataract is the most important reason of blinding which can be caused by the denaturalization of crystalline. Because of the complexiation of the cataractogenesis, we still have not known it clearly. Up to now, the study of cataractogenesis has become one of the hotspots in academia.To prevent and cure cataract, it is important to pay attention to the key link of the cataract coming on. For many years, researchers have study the cataractogenesis from many aspects. Filtration, identification, and clone related gene are always the hotspots. Our laboratory has ever found a spetial protein pot in 2-D gel which is only appeared in advanced stages of cataract and the concentration of it increased with the development of cataract. It is called X₃6-crystallin. Studies documented that this protein has three characteristics: It is only appeared in advanced stages of cataract and the concentration of it increased with the development of cataract while the other main members of lens crystalline(α,β,γ) are different from it. Westen blot resμlting shown this protein can not react with the antibody of α,β,γ-crystallin. This article is aimed at identification of X36 and the discussion of the relationship between X36 and cataract. In addition, we cloned and expressed another important protein: βB2 - crystallin which has a closed relationship with senile cataract and sugar cataract = Unfortunately, we couldn't clone and express X36 - crystallin because we haven't got it's accurate amino sequence. So, in the third part of this article, there are some station about the work on βB2 - crystallin.Part I Cataract Animal ModeTen Sprague-Dawley female rats (6-week-old) weighing 150-200 gramswere fed ground chow and injected 50% D-galactose solution every day in their abdominal cavity according to the dose of 10g/(kg ? d). At the mean time, the water they drink contains 10% D-galactose. Five to six weeks later, check their lens by cranny. After two months, one died, seven of them got visual lens cloudy and three of them got nucleal cataract.Part II Separation, Purification and Identification ofX36-crystallin.Water-soluble crystallins from the rats with nucleal cataract were extracted and separated by two-dimensional polyacrylamide gel electrophoresis. The gels without Comassize blue stining were electrotransfered to PVDF film for protein sequence analysis, the rest of them were stained by Comassize blue and then analysed by Mass Spectrum. Unfortunately, we haven't got it's accurate amino sequence because it's concentration is too low to test. But the Mass Spectrum results showed that X36-crystallin belongs to BBi-crystallin. What kind of modification occurred and what's the relationship between these modification and cataractogenesis are still unknown. According to the literature, we presume that there are three kinds of modification occoured. They are Truncation, Deamidation, and Glycation.Part HI Contents About BB2-Crystallin6B2-Crystallin is the major component of water-soluble 6-crystallin in aging lens and has a closed relationship with senile cataract and sugar cataract. It is essential to clone and express X36- and BB2-Crystallin for study of the interaction between them. Because we haven't got accurate amino sequence of X36, so we first cloned, expressed and purified 6B2-Crystallin. The constructed recombinant 6B2-pGEX plasmid had been identified by restriction...